Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour

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These microbeads are suggested to be nontoxic, biodegradable carriers conjugated to various antibodies. Isolation of cells through positive selection involves the attachment of antibody conjugated microbeads to the cells of interest, followed by their isolation in the presence of a strong magnetic field to obtain higher purity. Negative selection involves attachment of microbead conjugated antibodies to all other cell populations except the cells of interest, which remain untagged. In the present study, we compared the two methods for their effect on functional and immunophenotypic behavior of isolated CD14+ monocytes. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from blood collected from healthy volunteers by density gradient centrifugation. Human blood derived monocytes were isolated through positive selection and negative selection, making use of the appropriate monocyte isolation kit. Monocytes were then stimulated with lipopolysaccharide (LPS) and their activation and proliferation capacity were examined. The degradation or dissociation of cell-bound microbeads was also investigated. Results: We observed an impaired LPS sensitivity as well as poor activation and proliferation capacity upon stimulation by LPS in positively sorted CD14+ monocytes as compared to negatively sorted CD14+ monocytes. The attached microbeads did not degrade and remained attached to the cells even after 6 days of culture. Conclusions: Our results suggest that positively sorted CD14+ cells exhibit hampered functionality and may result in inaccurate analysis and observations in downstream applications. However, these cells can be used for immediate analytical procedures." } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/6-2045", "name": "Monocytes isolated by positive and negative magnetic sorting techniques..." } } ] } Home Browse Monocytes isolated by positive and negative magnetic sorting techniques... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Bhattacharjee J, Das B, Mishra A et al. Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour [version 3; peer review: 2 approved] . F1000Research 2018, 6 :2045 ( https://doi.org/10.12688/f1000research.12802.3 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Research Article Revised Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour [version 3; peer review: 2 approved] Jashdeep Bhattacharjee 1,2 , Barun Das 2 , Alaknanda Mishra 2 , Preeti Sahay 2 , Pramod Upadhyay https://orcid.org/0000-0002-5560-822X 2 Jashdeep Bhattacharjee 1,2 , Barun Das 2 , [...] Alaknanda Mishra 2 , Preeti Sahay 2 , Pramod Upadhyay https://orcid.org/0000-0002-5560-822X 2 PUBLISHED 28 Mar 2018 Author details Author details 1 Division of Gastroenterology, Hepatology and Nutrition, Children's Hospital Los Angeles, Los Angeles, CA, USA 2 National Institute of Immunology, New Delhi, India Jashdeep Bhattacharjee Roles: Conceptualization, Investigation, Methodology, Writing – Original Draft Preparation Barun Das Roles: Investigation, Methodology, Writing – Original Draft Preparation Alaknanda Mishra Roles: Investigation, Methodology, Writing – Review & Editing Preeti Sahay Roles: Methodology, Writing – Original Draft Preparation Pramod Upadhyay Roles: Conceptualization, Funding Acquisition, Investigation, Methodology, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS Abstract Background : Magnetic sorting of cells, based on microbead conjugated antibodies (Abs), employs positive as well as negative immunomagnetic separation methods, for isolation of a specific cell population. These microbeads are suggested to be nontoxic, biodegradable carriers conjugated to various antibodies. Isolation of cells through positive selection involves the attachment of antibody conjugated microbeads to the cells of interest, followed by their isolation in the presence of a strong magnetic field to obtain higher purity. Negative selection involves attachment of microbead conjugated antibodies to all other cell populations except the cells of interest, which remain untagged. In the present study, we compared the two methods for their effect on functional and immunophenotypic behavior of isolated CD14+ monocytes. Methods : Peripheral blood mononuclear cells (PBMCs) were isolated from blood collected from healthy volunteers by density gradient centrifugation. Human blood derived monocytes were isolated through positive selection and negative selection, making use of the appropriate monocyte isolation kit. Monocytes were then stimulated with lipopolysaccharide (LPS) and their activation and proliferation capacity were examined. The degradation or dissociation of cell-bound microbeads was also investigated. Results : We observed an impaired LPS sensitivity as well as poor activation and proliferation capacity upon stimulation by LPS in positively sorted CD14+ monocytes as compared to negatively sorted CD14+ monocytes. The attached microbeads did not degrade and remained attached to the cells even after 6 days of culture. Conclusions : Our results suggest that positively sorted CD14+ cells exhibit hampered functionality and may result in inaccurate analysis and observations in downstream applications. However, these cells can be used for immediate analytical procedures. READ ALL READ LESS Keywords immune-magnetic cell sorting, lipopolysaccharide sensitivity, CD14+ve monocytes Corresponding Author(s) Pramod Upadhyay ( [email protected] ) Close Corresponding author: Pramod Upadhyay Competing interests: No competing interests were disclosed. Grant information: This work has been supported by the core grant received from the Department of Biotechnology, Government of India, to the National Institute of Immunology, New Delhi. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2018 Bhattacharjee J et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication). How to cite: Bhattacharjee J, Das B, Mishra A et al. Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour [version 3; peer review: 2 approved] . F1000Research 2018, 6 :2045 ( https://doi.org/10.12688/f1000research.12802.3 ) First published: 23 Nov 2017, 6 :2045 ( https://doi.org/10.12688/f1000research.12802.1 ) Latest published: 28 Mar 2018, 6 :2045 ( https://doi.org/10.12688/f1000research.12802.3 ) Revised Amendments from Version 2 In the second revision of the manuscript following changes have been made. 1. A couple of sentences have been rewritten in the Introduction section to bring clarity on 'how positive magnetic sorting of CD14 may not be expected to trigger any signal transduction pathways'. 2. A typo has been corrected the Results section; sub section “Degradation of microbeads”. In the second revision of the manuscript following changes have been made. 1. A couple of sentences have been rewritten in the Introduction section to bring clarity on 'how positive magnetic sorting of CD14 may not be expected to trigger any signal transduction pathways'. 2. A typo has been corrected the Results section; sub section “Degradation of microbeads”. See the authors' detailed response to the review by Aniruddha Roy See the authors' detailed response to the review by Nadejda Beliakova-Bethell READ REVIEWER RESPONSES Introduction Magnetic sorting is a common technique used to obtain a highly pure population of cells of interest from a mixed population of cells, making use of microbead conjugated antibodies against the cell surface antigen. Positive sorting involves the tagging of cells with magnetic microbead conjugated antibodies, followed by isolation of the labeled cells by placing them in a magnetic field. After positive sorting, cells that have microbead conjugated antibodies on their surface can be conveniently analyzed using flow cytometry ( Miltenyi et al ., 1990 ; Pei et al ., 1998 ). Negative sorting involves the labeling of all cells, except the cells of interest, by incubating them in a cocktail of magnetic microbead conjugated antibodies and subsequently removing them by placing them in a magnetic field. Cluster of differentiation 14 (CD14) are specific markers used to identify monocytic populations, and they act as a coreceptors for LPS ( Guha & Mackman, 2001 ). Since CD14 lacks a cytoplasmatic domain, the positive magnetic sorting of CD14 may not be expected to trigger any signal transduction pathways or alter its functionality. However it has been demonstrated that anti-CD14 antibody reduces LPS responsiveness of monocytes ( Kim & Kim, 2014 ). We investigated the immunophenotypic behaviour and molecular characteristics of monocytes after both positive and negative sorting, by analyzing their response and proliferation to stimuli like LPS. The biodegradation profile of the attached microbeads from the CD14+ cells was also investigated. Methods Ethical statement The investigation was approved (project serial number: IHEC/#52/10) by the Institutional human ethics committee of the National Institute of Immunology, New Delhi-67, India. Isolation of PBMCs Experiments were performed at the National Institute of Immunology, New Delhi. 20 ml of peripheral blood was collected from five healthy volunteers aged between 25–30 years, after obtaining their written informed consent. Blood was collected more than once from some of the volunteers and there was a minimum gap of three months between two successive sample collections. The peripheral blood mononuclear cells (PBMCs) were isolated from blood by density gradient centrifugation using HiSep TM LSM 1077 (Himedia, Mumbai; India). The obtained PBMCs were washed thrice with Dulbecco’s phosphate buffered saline (Himedia, Mumbai; India) and counted using the trypan blue dye exclusion method with a hemocytometer (Rohem Industries Pvt Ltd, India). Isolation of monocytes by magnetic activated cell sorting Human blood derived monocytes were sorted using anti-human CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany), as per manufacturer’s protocol. Similarly, monocytes were isolated by negative sorting using the monocyte isolation Kit II (Miltenyi Biotec, Bergisch Gladbach; Germany) according to manufacturer’s protocol. Cell culture and antibody dissociation assay The positively sorted CD14 positive cells were re-suspended in RPMI 1640 medium (Himedia, Mumbai; India) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Beit-Haemek Israel) and 1X antibiotic-antimycotic solution containing streptomycin sulphate, penicillin and amphotericin-B (Himedia, Mumbai; India), and plated at a density of 4 × 10 6 cells per well in 6 well low adherence plates (Corning, Tewksbury; USA). The cells were periodically harvested by gentle scrapping and passed through a magnetic column. Cells with and without bound microbeads (obtained in the flowthrough) were counted using a hemocytometer (Rohem Industries Pvt Ltd, India). LPS stimulation of sorted monocytes Monocytes separated either by positive or negative selections were re-suspended in RPMI 1640 media supplemented with 10% FBS and 1X antibiotic-antimycotic solution. 1 million cells/ well were plated in a 24 well cell culture plate (Corning, Tewksbury, USA) and placed in a humidified CO 2 incubator (ShelLab, Cornelius; USA) at 5% CO 2 / 37°C for 24 hours. The cells were examined for adherence and thereafter stimulated with 1 ml complete RPMI media containing 500ng/ml of LPS (Sigma, St. Louis; USA). Fresh media containing 500ng/ml of LPS was replaced at each time point (8h, 16h, 24h) for supernatant collection. Cytometric bead array The supernatants collected at various time points were analysed for the presence of pro- and anti-inflammatory cytokines; IL-8, IL-10, TGF-β1 and RANTES, using cytometric bead array (CBA) Soluble Protein Flex Set (BD Biosciences, San Jose; USA) as per manufacturer’s protocol. Equal volumes of five independent samples were pooled and undiluted samples were analysed. The data was recorded using BD FACSVerse (BD Biosciences, San Jose; USA) and was analysed using FCAP Array software v3.0 (BD Biosciences, San Jose; USA). The assay samples were appropriately diluted to match the detection range of the CBA kit. Live cell imaging The magnetic sorted monocytes were plated at a density of 1 million cells per well in a 24 well plate. After allowing the cells to adhere for 24 hours, RPMI 1640 media supplemented with 10% FBS and 1% antibiotic-antimycotic solution containing 500 ng/ml of LPS was added to the respective wells. The culture plates were then placed in Cell-IQ (CM Technologies, Tampere; Finland) and specific fields were focussed using 10X objective magnification. Time lapse microscopy was performed and analysed for 76 hours at 30 minutes interval using live cell imaging and software (Cell IQ Analyser, Finland). Results Activation and proliferation of sorted CD14+ monocytes Sorted monocytes incubated with LPS were examined for secretion of various pro- and anti-inflammatory cytokines. The levels of IL-8, IL-10, TGF-β1 and RANTES at different time points after positively and negatively isolated CD14+ monocytes were incubated with LPS were analyzed. The secretion of IL-8 was observed to be at its maximum at 8 hours in negatively sorted monocytes and at 16 hours in positively sorted monocytes ( Figure 1A ). The IL-8 level in negatively sorted CD14+ cells was 6 times higher than positively sorted CD14+ cells. A similar pattern was observed for secretion of RANTES ( Figure 1C ) and TGF-β1 ( Figure 1D ), though the differences were not very pronounced. It was of significance to observe the reversed pattern for an anti-inflammatory cytokine, IL-10 ( Figure 1B ). Figure 1. The levels of IL-8, IL-10, TGF-β1 and RANTES in 5 pooled samples at different time points after positively and negatively isolated CD14+ monocytes were stimulated with LPS. Greater secretion of the pro-inflammatory cytokines in negatively sorted CD14+ monocytes upon activation was observed, during proliferation of activated monocytes. Figure 2 shows the variation in the average number of cells per field (487μm×364μm) for positively and negatively sorted cells. Further, the progression of proliferation is shown in Video V1 for negatively sorted cells and Video V2 for positively sorted cells. Alongside Figure 2 , the videos show that negatively sorted cells proliferated rapidly and extensively upon activation, and the maximum number of cells was reached after 16 hours. However, there was no clearly defined time point of maximum number of cells reached by positively sorted cells. Figure 2. The changes in the average number of cells per field (487μmX364μm) over time, upon stimulation by LPS in positively and negatively sorted cells. Degradation of microbeads To examine the degradation of microbeads, PBMCs were labeled with anti-human CD14 antibody conjugated microbeads and sorted under a magnetic field. The sorted cells were maintained in a culture and the numbers of cells with and without microbeads were counted via flow cytometric analysis. Results of three independent samples are shown in Figure 3 . Figure 3. Variation in the percentages of CD14+ cells which do not have bound anti-CD14 microbeads when they were cultured for several days. These results show the variation in percentage of cells collected in the ‘flow through’ of column placed in magnetic field. These were the cells from which the microbeads were either degraded or detached and cells were without microbeads. In all the three cultures we examined, the percentage of cells without microbeads was different but there was hardly any change in these percentages when the culture was continued for 6 days. This suggests that in typical culture conditions the Ab-microbeads remain bound with cells for many days. Time IL8(Neg) IL8(Pos) TGF(Neg) TGF(Pos) RANT(Neg) RANT(Pos) IL10(Neg) IL10(Pos) 8 6.40E+07 101014.87 103.89 70.45 100.51 30.59 490.07 1753.48 16 364584.34 9.84E+06 96.88 55.68 19.48 5.52 84.21 422.81 24 27205.78 2.64E+06 87.71 64.28 9.46 0 31.42 66.55 Dataset 1. Raw data corresponding to the results shown in Figure 1. A PositiveSor NegativeSor TimeHours 262 1821.07407 1800.52778 0.07278 1871 1775.74074 1803.83333 0.51972 3360 1735.40741 1875.41667 0.93333 4856 1712.59259 1998.69444 1.34889 6345 1730.03704 2006 1.7625 7829 1721 1941.16667 2.17472 9310 1785.48148 1916.22222 2.58611 10795 1826.14815 1903.52778 2.99861 12279 1868.88889 1904.80556 3.41083 13764 1853.77778 1903.69444 3.82333 15249 1872.33333 1898.75 4.23583 16733 1890.2963 1884.25 4.64806 18221 1895.92593 1879.25 5.06139 19706 1902.25926 1863.72222 5.47389 21192 1931.44444 1865.88889 5.88667 22678 1922.66667 1840.55556 6.29944 24161 1948.14815 1867.13889 6.71139 25650 1976.74074 1856.88889 7.125 27133 1992.2963 1851.08333 7.53694 28618 1995.07407 1855.86111 7.94944 30100 2016.37037 1840.58333 8.36111 31587 2044.37037 1848.02778 8.77417 33068 2060.66667 1857.11111 9.18556 34555 2073.77778 1829.05556 9.59861 36045 2087.92593 1834.5 10.0125 37534 2135.14815 1816.72222 10.42611 39020 2172.25926 1836.83333 10.83889 40501 2171.07407 1825.5 11.25028 41985 2178.77778 1804.55556 11.6625 43473 2230.66667 1824.75 12.07583 44957 2246.25926 1841.94444 12.48806 46443 2249.11111 1822.52778 12.90083 47931 2299.92593 1832.69444 13.31417 49425 2325.51852 1824.69444 13.72917 50912 2345.2963 1825.38889 14.14222 52399 2367.62963 1850.08333 14.55528 53882 2393.62963 1831.41667 14.96722 55367 2425.62963 1839.44444 15.37972 56857 2443 1842.66667 15.79361 58344 2447.40741 1838.27778 16.20667 59830 2430 1847.19444 16.61944 61312 2492.51852 1844.27778 17.03111 62799 2504.51852 1842.66667 17.44417 64286 2530.18518 1849.08333 17.85722 65771 2499.59259 1855.94444 18.26972 67254 2517.88889 1882.61111 18.68167 68736 2525.77778 1878.97222 19.09333 70233 2562.18518 1884.61111 19.50917 71721 2597.88889 1867.27778 19.9225 73203 2593.14815 1853.52778 20.33417 74690 2578.92593 1865.52778 20.74722 76182 2600.14815 1864.91667 21.16167 77670 2604.18518 1857.02778 21.575 79153 2598.18518 1865.5 21.98694 80638 2641.44444 1876.27778 22.39944 82122 2650.22222 1860.75 22.81167 83611 2642.03704 1877.47222 23.22528 85094 2661.77778 1886.86111 23.63722 86583 2642.40741 1850.69444 24.05083 88077 2675.37037 1877.44444 24.46583 89568 2661.92593 1871.25 24.88 91063 2621.07407 1860.33333 25.29528 92551 2655.55556 1864.27778 25.70861 94047 2617.03704 1856.27778 26.12417 95534 2633.03704 1879.33333 26.53722 97032 2630.62963 1856.94444 26.95333 98521 2630.81482 1878.02778 27.36694 100013 2615.22222 1858.55556 27.78139 101505 2600.22222 1881.61111 28.19583 102991 2588.40741 1879.80556 28.60861 104483 2581.77778 1845.41667 29.02306 105971 2609.14815 1876.36111 29.43639 107460 2588.33333 1862.61111 29.85 108946 2595.51852 1900.47222 30.26278 110439 2598.51852 1868.52778 30.6775 111924 2602.14815 1855.88889 31.09 113407 2588.44444 1878.19444 31.50194 114898 2575.7037 1870.05556 31.91611 116388 2562.2963 1878.77778 32.33 117875 2559.7037 1868.44444 32.74306 119377 2564.92593 1838.05556 33.16028 120874 2535.40741 1860.66667 33.57611 122371 2529.33333 1834.77778 33.99194 123862 2523.74074 1876.33333 34.40611 125351 2529.92593 1881.80556 34.81972 126844 2503.51852 1814.19444 35.23444 128343 2505.85185 1853.13889 35.65083 129838 2466.40741 1820.86111 36.06611 131332 2460.59259 1814.88889 36.48111 132826 2459.59259 1849.77778 36.89611 134324 2463.85185 1838.58333 37.31222 135813 2440 1863.05556 37.72583 137305 2438.25926 1863.13889 38.14028 138798 2418.48148 1825.75 38.555 140294 2405.44444 1822.36111 38.97056 141791 2393 1806.38889 39.38639 143285 2375.07407 1833.27778 39.80139 144773 2371.14815 1809.13889 40.21472 146258 2312.62963 1812.27778 40.62722 147748 2314.11111 1751.5 41.04111 149240 2328.66667 1819.05556 41.45556 150729 2337.14815 1854.72222 41.86917 152223 2320.81482 1808.80556 42.28417 153714 2330.11111 1857.72222 42.69833 155204 2314.92593 1824.55556 43.11222 156693 2296.44444 1776.33333 43.52583 158184 2302.81482 1827.38889 43.94 159676 2272.55556 1793.02778 44.35444 161162 2266.07407 1792.02778 44.76722 162652 2268.48148 1786.5 45.18111 164143 2263.37037 1783.02778 45.59528 165632 2249.85185 1749.55556 46.00889 167119 2257.14815 1808.47222 46.42194 168613 2271.88889 1788.08333 46.83694 170105 2226.2963 1763.5 47.25139 171592 2247.74074 1781.86111 47.66444 173085 2222.2963 1743.02778 48.07917 174580 2213.59259 1788 48.49444 176072 2208.51852 1800.33333 48.90889 177560 2176.03704 1801.72222 49.32222 179051 2170.14815 1734.77778 49.73639 180544 2185.03704 1724.05556 50.15111 182041 2124.03704 1701.69444 50.56694 183529 2152.55556 1730.47222 50.98028 185027 2138.11111 1707.11111 51.39639 186520 2119.03704 1742.05556 51.81111 188011 2115.25926 1774.27778 52.22528 189507 2092.37037 1720.05556 52.64083 191004 2116.18518 1737.58333 53.05667 192497 2075.11111 1688.66667 53.47139 193992 2077.18518 1747.11111 53.88667 195486 2066.22222 1763.97222 54.30167 196986 2057.11111 1699.22222 54.71833 198484 2038.88889 1675.27778 55.13444 199982 2065.22222 1708.19444 55.55056 201468 2028.40741 1688.91667 55.96333 202965 2027 1679.72222 56.37917 204459 2014.85185 1711.19444 56.79417 205952 2019.66667 1686.83333 57.20889 207456 2007.48148 1663.27778 57.62667 208958 1974.2963 1640.30556 58.04389 210459 1960.07407 1606.5 58.46083 211955 1969.11111 1676.63889 58.87639 213455 1940.03704 1627.16667 59.29306 214958 1924 1650.02778 59.71056 216461 1919.74074 1640.38889 60.12806 217960 1876 1566.5 60.54444 219457 1890.85185 1601.47222 60.96028 220956 1881.25926 1600.08333 61.37667 222449 1848.92593 1601.19444 61.79139 223941 1878.92593 1617.27778 62.20583 225438 1849.22222 1592.05556 62.62167 226935 1816.81482 1586.41667 63.0375 228432 1813.40741 1616.75 63.45333 229929 1814.59259 1576.33333 63.86917 231424 1820.55556 1604.97222 64.28444 232920 1818.77778 1583.25 64.7 234421 1800.66667 1532.11111 65.11694 235915 1800.33333 1559.44444 65.53194 237409 1798.03704 1550.94444 65.94694 238910 1780.48148 1583.86111 66.36389 240408 1763.03704 1535.5 66.78 241906 1747.25926 1530.36111 67.19611 243401 1753.22222 1580.80556 67.61139 244888 1716.11111 1533 68.02444 246383 1710.92593 1553.75 68.43972 247882 1705.25926 1532.44444 68.85611 249380 1698.59259 1519.41667 69.27222 250875 1692.59259 1515 69.6875 252368 1676.25926 1485.80556 70.10222 253861 1670.7037 1464.66667 70.51694 255357 1670.2963 1480.77778 70.9325 256847 1649.14815 1510.97222 71.34639 258345 1625.48148 1498 71.7625 259834 1611.62963 1452.77778 72.17611 261329 1611.2963 1450.44444 72.59139 262829 1606.44444 1390.27778 73.00806 264320 1617.03704 1411.27778 73.42222 265812 1612.03704 1429.33333 73.83667 267298 1598.40741 1402.44444 74.24944 268790 1598.11111 1420.52778 74.66389 270280 1603.88889 1412.36111 75.07778 271778 1567.96296 1369.66667 75.49389 273275 1569.37037 1416.61111 75.90972 274763 1559.22222 1416.63889 76.32306 Dataset 2. Raw data corresponding to the results shown in Figure 2. A Day2 Day4 Day6 E F G H Sample1 20 17 18 Sample 2 5 4.6 7.4 Dataset 3. Raw data corresponding to the results shown in Figure 3. Discussion Magnetic cell sorting for the separation of large numbers of cells according to specific cell surface markers is a technique that is commonly used. It is a common notion that magnetic beads are biodegradable, do not activate cells and do not affect downstream application. We have however observed that the activation and proliferation of positively sorted CD14+ cells is impaired compared to the negatively sorted cells. The activation of monocytes by LPS is known to occur through surface CD14, which is an LPS sensing receptor. Surface CD14 plays a crucial role; it binds and transfers LPS to the surface via TLR4:MD2 complex to enable its recognition. The LPS stimulation of monocytes activates several intracellular signaling pathways which in turn activates a variety of transcription factors ultimately leading to induction of many genes encoding inflammatory cytokines ( Guha & Mackman, 2001 ). In short, CD14 is involved in the LPS-induced release of IL-8, which is an important pro-inflammatory cytokine ( He et al ., 2013 ). After positively sorting CD14+ monocytes from PBMCs, the surface CD14 molecules on monocytes are blocked by anti-CD14 microbeads and these CD14+ surface sites can no longer mediate the stimulation by LPS and the positively sorted CD14+ monocytes may show impaired stimulation by LPS. In two experiments, identical numbers of CD14+ monocytes isolated by positive and negative sorting were stimulated with LPS and their activation and proliferation was monitored. Figure 1 shows that upon stimulation by LPS the negatively sorted CD14+ monocytes secreted enormous amount of IL-8 almost instantaneously and they exhibited acute proliferation ( Figure 2 ). This is in accordance with the observation that IL-8 transcript is highly expressed in LPS-stimulated monocytes ( Standiford et al ., 1992 ; Suzuki et al ., 2000 ). The positively sorted CD14+ monocytes responded only after 24 hours, and their level of stimulation was impaired and the cells did not proliferate. This delayed and reduced stimulation by LPS is due to the CD14 independent receptors which function to direct LPS mediated cytokine secretion under conditions where the CD14 dependent pathway is blocked or non-functional ( Lynn et al ., 1993 ). There are a few LPS-associated cell surface proteins which are distinct from CD14 and these surface proteins too can bind TLRs to initiate a response ( Triantafilou et al ., 2001 ). Our results suggest that the density of these surface proteins is low compared to CD14 as lesser stimulation was observed when CD14 surface groups were blocked by Abs and secondly, the delayed stimulation indicate that most likely a different pathway was followed for their activation. Further, the secretion of somewhat higher amount of IL-10 by positively sorted CD14+ monocytes only suggest the absence of highly inflammatory conditions upon LPS activation. The levels of RANTES and TGF-β1 also indicate that the LPS activation of monocytes via CD14 independent receptors produces unique results which could be very different from common experimental situation. In one such related report, the, human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated to macrophages ( Neu et al ., 2013 ). The phagocytosis of Listeria monocytogenes (Lm) by GM-CSF-derived macrophages (GM-M) was markedly influenced by the method used for isolation of monocytes. The GM-M derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-M generated from positively selected monocytes displayed high phagocytosis of Lm. The paper concludes that macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages. In yet another report ( Elkord et al ., 2005 ) it was demonstrated that the human dendritic cells generated from positively isolated monocytes by anti-CD14-coated microbeads show impaired induction by LPS. Beliakova-Bethell et al. ( Beliakova-Bethell et al ., 2014 ) examined the gene expression profiles of CD8+ T cells, B cells and monocytes isolated using positive selection, negative selection and FACS. They concluded that gene expression signatures were more similar between cells isolated by negative selection and FACS compared to cells isolated by positive selection. These findings were made on cells immediately after isolation and our findings establish the long-term effects of positive and negative isolation methods. In these reports it was not investigated further why the positively isolated monocytes were not suitable and had poor cytokines production upon stimulation. Our data suggests that in these experiments, the positively isolated monocytes were tagged permanently with anti-CD14 molecules attached with microbeads. The positively isolated CD14+ monocytes are identical with monocytes whose surface CD14 molecules have been blocked by Abs and these monocytes are known to behave differently ( Delirezh et al ., 2013 ; Elkord et al ., 2005 ; Kim & Kim, 2014 ). An alternate strategy to positively isolate monocytes could be by using anti-CD33 coated beads instead of anti-CD14 coated beads. The monocytes isolated using this approach too most likely will result in impaired response upon LPS stimulation. It has been reported that CD33 and CD14 are colocalized on cell surface and when monocyte-derived immature dendritic cells were stimulated with LPS in the presence of CD33 antibody, the production of IL-12 and phosphorylation of NF-κB decreased significantly ( Ishida et al ., 2014 ). The monocytes culture in the presence of anti-CD33 and the positively isolated monocytes using anti-CD33 coated beads, both have antibody bound CD33, and both are likely to respond in a similar manner. In this context, diverse outcome could be observed when cells positively isolated by antibody bound microbeads were used for extended culture work ( Govers et al ., 2012 ; Greish et al ., 2012 ; Lapenna et al ., 2013 ; Meinhardt et al ., 2012 ). There are two important findings from these experiments; positively isolated CD14+ monocytes have impaired LPS sensitivity and magnetic beads used in positive isolation do not degrade within days. These conclusions suggest that for most experiments, positively isolated cells are usable for analysis purpose only and should not be used for any further culture experiments. Data availability Dataset 1: Raw data corresponding to the results shown in Figure 1 . DOI, 10.5256/f1000research.12802.d188477 ( Bhattacharjee et al ., 2017a ) Dataset 2: Raw data corresponding to the results shown in Figure 2 . DOI, 10.5256/f1000research.12802.d182357 ( Bhattacharjee et al ., 2017b ) Dataset 3: Raw data corresponding to the results shown in Figure 3 . DOI, 10.5256/f1000research.12802.d182358 ( Bhattacharjee et al ., 2017c ) Competing interests No competing interests were disclosed. Grant information This work has been supported by the core grant received from the Department of Biotechnology, Government of India, to the National Institute of Immunology, New Delhi. Author details This work was carried out at: National Institute of Immunology, New Delhi, India. The first author, Jashdeep Bhattacharjee, has now moved to: Division of Gastroenterology, Hepatology and Nutrition, Children’s Hospital Los Angeles. Supplementary material Supplementary Video V1: The progression of proliferation after stimulation by LPS in negatively sorted CD14+ monocytes. Click here to access the data . Supplementary Video V2: The progression of proliferation after stimulation by LPS in positively sorted CD14+ monocytes. Click here to access the data . Faculty Opinions recommended References Beliakova-Bethell N, Massanella M, White C, et al. : The effect of cell subset isolation method on gene expression in leukocytes. Cytometry A. 2014; 85 (1): 94–104. PubMed Abstract | Publisher Full Text | Free Full Text Bhattacharjee J, Das B, Mishra A, et al. : Dataset 1 in: Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour. F1000Research. 2017a. Data Source Bhattacharjee J, Das B, Mishra A, et al. : Dataset 2 in: Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour. F1000Research. 2017b. Data Source Bhattacharjee J, Das B, Mishra A, et al. : Dataset 3 in: Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour. F1000Research. 2017c. Data Source Delirezh N, Shojaeefar E, Parvin P, et al. : Comparison the effects of two monocyte isolation methods, plastic adherence and magnetic activated cell sorting methods, on phagocytic activity of generated dendritic cells. 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PubMed Abstract | Publisher Full Text Comments on this article Comments (0) Version 3 VERSION 3 PUBLISHED 23 Nov 2017 ADD YOUR COMMENT Comment Author details Author details 1 Division of Gastroenterology, Hepatology and Nutrition, Children's Hospital Los Angeles, Los Angeles, CA, USA 2 National Institute of Immunology, New Delhi, India Jashdeep Bhattacharjee Roles: Conceptualization, Investigation, Methodology, Writing – Original Draft Preparation Barun Das Roles: Investigation, Methodology, Writing – Original Draft Preparation Alaknanda Mishra Roles: Investigation, Methodology, Writing – Review & Editing Preeti Sahay Roles: Methodology, Writing – Original Draft Preparation Pramod Upadhyay Roles: Conceptualization, Funding Acquisition, Investigation, Methodology, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information This work has been supported by the core grant received from the Department of Biotechnology, Government of India, to the National Institute of Immunology, New Delhi. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (3) version 3 Revised Published: 28 Mar 2018, 6:2045 https://doi.org/10.12688/f1000research.12802.3 version 2 Revised Published: 22 Dec 2017, 6:2045 https://doi.org/10.12688/f1000research.12802.2 version 1 Published: 23 Nov 2017, 6:2045 https://doi.org/10.12688/f1000research.12802.1 Copyright © 2018 Bhattacharjee J et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication). Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Bhattacharjee J, Das B, Mishra A et al. Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour [version 3; peer review: 2 approved] . F1000Research 2018, 6 :2045 ( https://doi.org/10.12688/f1000research.12802.3 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 2 VERSION 2 PUBLISHED 22 Dec 2017 Revised Views 0 Cite How to cite this report: Roy A. Reviewer Report For: Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour [version 3; peer review: 2 approved] . F1000Research 2018, 6 :2045 ( https://doi.org/10.5256/f1000research.14717.r31885 ) The direct URL for this report is: https://f1000research.com/articles/6-2045/v2#referee-response-31885 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 23 Mar 2018 Aniruddha Roy , Department of Pharmacy, Birla Institute of Technology & Science, Pilani (BITS), Pilani, India Approved VIEWS 0 https://doi.org/10.5256/f1000research.14717.r31885 In the current manuscript, the authors have investigated the impact of positive selection and negative selection on the activation, function, and proliferation of monocytes. It is an important study as many researchers use these techniques interchangeably, greatly impacting the final outcome ... Continue reading READ ALL In the current manuscript, the authors have investigated the impact of positive selection and negative selection on the activation, function, and proliferation of monocytes. It is an important study as many researchers use these techniques interchangeably, greatly impacting the final outcome of the study and erroneous conclusion. Though the current study has used only a few experiments to draw the conclusion, their study design was neat. The authors may clarify some minor points to make the manuscript better: In the introduction section, the authors have written: "CD14 lacks a cytoplasmatic domain and such cells are reported to function in a restricted manner" - a brief explanation of what is meant by restricted manner would be helpful. The sentence on page number 5 reads: 'These were the cells from which the microbeads were either degraded or " detected " and cells were without microbeads.' : Probably instead of detected, it will be detached. Is the size of the beads known? Can there be a size-dependent effect due to steric hindrance of the CD14 bound beads to the TLR4? If only antibody is used, can we see the same effect? The main function of CD14 in LPS signaling in monocytes is it binds with LPS binding protein (LBP). The role of LBP appears to be that of aiding LPS to dock at the LPS receptor complex by initially binding LPS and then forming a ternary complex with CD14, thus enabling LPS to be transferred to the LPS receptor complex composed of TLR4 and MD-2. As in these in vitro studies, LBP has not been used, probably CD14 may not directly affect the LPS binding with the TLR4 receptor. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Roy A. Reviewer Report For: Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour [version 3; peer review: 2 approved] . F1000Research 2018, 6 :2045 ( https://doi.org/10.5256/f1000research.14717.r31885 ) The direct URL for this report is: https://f1000research.com/articles/6-2045/v2#referee-response-31885 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 28 Mar 2018 Pramod Upadhyay , National Institute of Immunology, New Delhi, India 28 Mar 2018 Author Response Thank you for providing us valuable comments. We have addressed all of them in the revised version of the manuscript. Specifically In the introduction section, the authors have ... Continue reading Thank you for providing us valuable comments. We have addressed all of them in the revised version of the manuscript. Specifically In the introduction section, the authors have written: "CD14 lacks a cytoplasmatic domain and such cells are reported to function in a restricted manner" - a brief explanation of what is meant by restricted manner would be helpful. It was an inadvertent mistake to write an incomplete sentence. This has been corrected in the revised manuscript. The sentence on page number 5 reads: 'These were the cells from which the microbeads were either degraded or "detected" and cells were without microbeads.' : Probably instead of detected, it will be detached. The correction has been made in the revised manuscript. Is the size of the beads known? Can there be a size-dependent effect due to steric hindrance of the CD14 bound beads to the TLR4? If only antibody is used, can we see the same effect? The main function of CD14 in LPS signaling in monocytes is it binds with LPS binding protein (LBP). The role of LBP appears to be that of aiding LPS to dock at the LPS receptor complex by initially binding LPS and then forming a ternary complex with CD14, thus enabling LPS to be transferred to the LPS receptor complex composed of TLR4 and MD-2. As in these in vitro studies, LBP has not been used, probably CD14 may not directly affect the LPS binding with the TLR4 receptor. Nano meter size beads are used is cells isolation and these are unlikely to create significant steric hindrance. The main point is that once Ab-conjugated beads get bound to the antigen cell surface (CD14 in present case); it is bound to influence the expression, transcription and translation machinery inside the cell. And secondly, as a result of conjugation of cell surface CD14 molecule with Ab-conjugated bead in cells isolation step, the cell surface CD14 molecule would no longer be available for LBP to initiate the activation cascade. This has been elaborated in the Introduction section of the revised manuscript. Thank you for providing us valuable comments. We have addressed all of them in the revised version of the manuscript. Specifically In the introduction section, the authors have written: "CD14 lacks a cytoplasmatic domain and such cells are reported to function in a restricted manner" - a brief explanation of what is meant by restricted manner would be helpful. It was an inadvertent mistake to write an incomplete sentence. This has been corrected in the revised manuscript. The sentence on page number 5 reads: 'These were the cells from which the microbeads were either degraded or "detected" and cells were without microbeads.' : Probably instead of detected, it will be detached. The correction has been made in the revised manuscript. Is the size of the beads known? Can there be a size-dependent effect due to steric hindrance of the CD14 bound beads to the TLR4? If only antibody is used, can we see the same effect? The main function of CD14 in LPS signaling in monocytes is it binds with LPS binding protein (LBP). The role of LBP appears to be that of aiding LPS to dock at the LPS receptor complex by initially binding LPS and then forming a ternary complex with CD14, thus enabling LPS to be transferred to the LPS receptor complex composed of TLR4 and MD-2. As in these in vitro studies, LBP has not been used, probably CD14 may not directly affect the LPS binding with the TLR4 receptor. Nano meter size beads are used is cells isolation and these are unlikely to create significant steric hindrance. The main point is that once Ab-conjugated beads get bound to the antigen cell surface (CD14 in present case); it is bound to influence the expression, transcription and translation machinery inside the cell. And secondly, as a result of conjugation of cell surface CD14 molecule with Ab-conjugated bead in cells isolation step, the cell surface CD14 molecule would no longer be available for LBP to initiate the activation cascade. This has been elaborated in the Introduction section of the revised manuscript. Competing Interests: There are competing interests. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 28 Mar 2018 Pramod Upadhyay , National Institute of Immunology, New Delhi, India 28 Mar 2018 Author Response Thank you for providing us valuable comments. We have addressed all of them in the revised version of the manuscript. Specifically In the introduction section, the authors have ... Continue reading Thank you for providing us valuable comments. We have addressed all of them in the revised version of the manuscript. Specifically In the introduction section, the authors have written: "CD14 lacks a cytoplasmatic domain and such cells are reported to function in a restricted manner" - a brief explanation of what is meant by restricted manner would be helpful. It was an inadvertent mistake to write an incomplete sentence. This has been corrected in the revised manuscript. The sentence on page number 5 reads: 'These were the cells from which the microbeads were either degraded or "detected" and cells were without microbeads.' : Probably instead of detected, it will be detached. The correction has been made in the revised manuscript. Is the size of the beads known? Can there be a size-dependent effect due to steric hindrance of the CD14 bound beads to the TLR4? If only antibody is used, can we see the same effect? The main function of CD14 in LPS signaling in monocytes is it binds with LPS binding protein (LBP). The role of LBP appears to be that of aiding LPS to dock at the LPS receptor complex by initially binding LPS and then forming a ternary complex with CD14, thus enabling LPS to be transferred to the LPS receptor complex composed of TLR4 and MD-2. As in these in vitro studies, LBP has not been used, probably CD14 may not directly affect the LPS binding with the TLR4 receptor. Nano meter size beads are used is cells isolation and these are unlikely to create significant steric hindrance. The main point is that once Ab-conjugated beads get bound to the antigen cell surface (CD14 in present case); it is bound to influence the expression, transcription and translation machinery inside the cell. And secondly, as a result of conjugation of cell surface CD14 molecule with Ab-conjugated bead in cells isolation step, the cell surface CD14 molecule would no longer be available for LBP to initiate the activation cascade. This has been elaborated in the Introduction section of the revised manuscript. Thank you for providing us valuable comments. We have addressed all of them in the revised version of the manuscript. Specifically In the introduction section, the authors have written: "CD14 lacks a cytoplasmatic domain and such cells are reported to function in a restricted manner" - a brief explanation of what is meant by restricted manner would be helpful. It was an inadvertent mistake to write an incomplete sentence. This has been corrected in the revised manuscript. The sentence on page number 5 reads: 'These were the cells from which the microbeads were either degraded or "detected" and cells were without microbeads.' : Probably instead of detected, it will be detached. The correction has been made in the revised manuscript. Is the size of the beads known? Can there be a size-dependent effect due to steric hindrance of the CD14 bound beads to the TLR4? If only antibody is used, can we see the same effect? The main function of CD14 in LPS signaling in monocytes is it binds with LPS binding protein (LBP). The role of LBP appears to be that of aiding LPS to dock at the LPS receptor complex by initially binding LPS and then forming a ternary complex with CD14, thus enabling LPS to be transferred to the LPS receptor complex composed of TLR4 and MD-2. As in these in vitro studies, LBP has not been used, probably CD14 may not directly affect the LPS binding with the TLR4 receptor. Nano meter size beads are used is cells isolation and these are unlikely to create significant steric hindrance. The main point is that once Ab-conjugated beads get bound to the antigen cell surface (CD14 in present case); it is bound to influence the expression, transcription and translation machinery inside the cell. And secondly, as a result of conjugation of cell surface CD14 molecule with Ab-conjugated bead in cells isolation step, the cell surface CD14 molecule would no longer be available for LBP to initiate the activation cascade. This has been elaborated in the Introduction section of the revised manuscript. Competing Interests: There are competing interests. Close Report a concern COMMENT ON THIS REPORT Version 1 VERSION 1 PUBLISHED 23 Nov 2017 Views 0 Cite How to cite this report: Beliakova-Bethell N. Reviewer Report For: Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour [version 3; peer review: 2 approved] . F1000Research 2018, 6 :2045 ( https://doi.org/10.5256/f1000research.13872.r28324 ) The direct URL for this report is: https://f1000research.com/articles/6-2045/v1#referee-response-28324 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 12 Dec 2017 Nadejda Beliakova-Bethell , VA San Diego Healthcare System, San Diego, CA, USA Approved VIEWS 0 https://doi.org/10.5256/f1000research.13872.r28324 This study assesses monocyte function following isolation of these cells from total peripheral blood mononuclear cells (PBMC) using positive or negative immunomagnetic selection. It addresses an important topic, because cell isolation procedure may affect cell function and skew conclusions obtained ... Continue reading READ ALL This study assesses monocyte function following isolation of these cells from total peripheral blood mononuclear cells (PBMC) using positive or negative immunomagnetic selection. It addresses an important topic, because cell isolation procedure may affect cell function and skew conclusions obtained from in vitro studies of immune cells isolated from blood. Despite a limited assessment of functions and isolation methods, this study should have an impact on selection of monocyte isolation protocol when these cells are intended for investigation of specific responses, which this study tested. The study design is appropriate and conclusions drawn are adequately supported by the results. The paper may be improved, however, by providing more details in methods, raw data and discussion, and by careful revision of citations as detailed below: Methods: Cytometic bead array: there are several cytometric bead arrays available for human samples (e.g. “human inflammatory cytokine kit”, “human chemokine kit”). Which one was used? Legend for Figure 1 indicates that 5 samples were pooled. It would be helpful to provide details on how the pooling was done (the entire samples, equal volumes, equal proportions?) and what dilutions were made for the assay. Live cell imaging: It appears that information in methods contradicts the data shown in Figure 2. According to the figure and supplied raw data, imaging was performed over a course of over 76 hours, while methods section states “48 hours”. Raw data: Data for Figure 1: it might be easier to understand the data if columns were labeled by cytokine and sample (e.g. IL8 negative selection, IL8 positive selection, etc. as opposed to IL8B, IL8C, etc.) Data for Figure 2: were 36 fields for each sample for each time point individually counted, as the information on the figure indicates? Raw data appears to have only the averages. Was the distribution of counts tight, or was there high variation? Including raw data for individual field counts might be more appropriate. Discussion: Another option to positively isolate monocytes is to use anti-CD33 instead of anti-CD14 coated beads. This may be more appropriate in studies that aim to measure response to LPS, when positive selection is preferred (e.g. in cases of limited sample from which sequential separation of different lymphocyte subsets is desired). It might be worthy to discuss this option. Citations: Several citations throughout the paper appear to be inaccurate; for example, in Introduction some citations on altered behavior of cells following microbead attachment: (1) Safarik and Safarikova is a method review, and Horgan et al. is a protocol; neither studies cell behavior; (2) Semple et al. observed no functional changes in limited tests they did; the difference that they report pertains to CD19+ vs CD19- cells, not positively isolated vs negatively isolated cells; (3) Fuchslin et al. labeled bacterial cells for their quantification in water; and Ribaut et al. labeled parasites for their isolation from infected blood for research purposes; the relevance of these two citations for the present study may be questionable. In Discussion, Adams et al. describe a special case of MACS for multitarget sort, and may not be appropriate for referencing commonality of magnetic sorting. While the results from the present study are consistent with gene expression studies by Beliakova-Bethell et al. in terms of effects of positive selection, the reference to Beliakova-Bethell et al. is made following description of Neu et al. and Elkord et al. studies, stating that the findings were similar. This is not accurate because Neu et al. and Elkord et al. performed functional assessment following cell separation, while Beliakova-Bethell et al. lysed the cells immediately after isolation and did not measure cell function. Instead of the statement of similarity of findings, it might be worth pointing out that positive selection affects monocytes the most both in the short term and in the long term. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Virology, immunology, cell separation and flow cytometry, analysis of gene expression I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Beliakova-Bethell N. Reviewer Report For: Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour [version 3; peer review: 2 approved] . F1000Research 2018, 6 :2045 ( https://doi.org/10.5256/f1000research.13872.r28324 ) The direct URL for this report is: https://f1000research.com/articles/6-2045/v1#referee-response-28324 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 22 Dec 2017 Pramod Upadhyay , National Institute of Immunology, New Delhi, India 22 Dec 2017 Author Response Thank you for providing us valuable comments. We have addressed all of them in the revised version of the manuscript. Specifically Methods: Cytometic bead array: there are several ... Continue reading Thank you for providing us valuable comments. We have addressed all of them in the revised version of the manuscript. Specifically Methods: Cytometic bead array: there are several cytometric bead arrays available for human samples (e.g. “human inflammatory cytokine kit”, “human chemokine kit”). Which one was used? Legend for Figure 1 indicates that 5 samples were pooled. It would be helpful to provide details on how the pooling was done (the entire samples, equal volumes, equal proportions?) and what dilutions were made for the assay. We used Cytometric Bead Array (CBA) Soluble Protein Flex Set and equal volumes of the samples were pooled. The CBA assay was performed on culture supernatants without any dilution. These details have been included in the revised manuscript. Live cell imaging: It appears that information in methods contradicts the data shown in Figure 2. According to the figure and supplied raw data, imaging was performed over a course of over 76 hours, while methods section states “48 hours”. Live cell imaging: The imaging was performed till 76 hours and it was a mistake to mention this as 48 hours in the method section, we thank you for pointing out this anomaly and this has been corrected in the revised manuscript. Raw data: Data for Figure 1: it might be easier to understand the data if columns were labeled by cytokine and sample (e.g. IL8 negative selection, IL8 positive selection, etc. as opposed to IL8B, IL8C, etc.) Suggested changes have been made. Data for Figure 2: were 36 fields for each sample for each time point individually counted, as the information on the figure indicates? Raw data appears to have only the averages. Was the distribution of counts tight, or was there high variation? Including raw data for individual field counts might be more appropriate . During the live cell imaging experiment images were logged and later cells were counted using the Cell-IQ Analyser software. The protocol for cell counting in the analysis software was assembled to finally provide the average number of cell count in the selected fields. The cell counts from individual fields of various images were embedded in the analysis software, thought it was possible to export these tables but due to very large number of data files we focussed only on the averages. Discussion: Another option to positively isolate monocytes is to use anti-CD33 instead of anti-CD14 coated beads. This may be more appropriate in studies that aim to measure response to LPS, when positive selection is preferred (e.g. in cases of limited sample from which sequential separation of different lymphocyte subsets is desired). It might be worthy to discuss this option. This is a good suggestion keeping in mind that the CD14 is an endogenous ligand for CD33 in monocyte-derived immature dendritic cells. It has been reported (Hiroshi Nakada et al.; J Biol Chem. 2014 Sep 5; 289(36): 25341–25350) that when monocyte-derived immature dendritic cells were stimulated with LPS in the presence of anti-CD33Ab, the production of IL-12 and phosphorylation of NF-κB decreased significantly. Most likely the positively isolated monocytes using CD33-Ab beads would behave in a similar manner. This possibility has been discussed in the Discussion section of the revised manuscript. Citations: Several citations throughout the paper appear to be inaccurate; for example, in Introduction some citations on altered behavior of cells following microbead attachment: We once again thank the reviewer for thoroughly examining the citations; the list of references has been appropriately revised. The differences between the findings of Beliakova-Bethell et al. and Neu et al. & Elkord et al. have been incorporated in the revised manuscript. Thank you for providing us valuable comments. We have addressed all of them in the revised version of the manuscript. Specifically Methods: Cytometic bead array: there are several cytometric bead arrays available for human samples (e.g. “human inflammatory cytokine kit”, “human chemokine kit”). Which one was used? Legend for Figure 1 indicates that 5 samples were pooled. It would be helpful to provide details on how the pooling was done (the entire samples, equal volumes, equal proportions?) and what dilutions were made for the assay. We used Cytometric Bead Array (CBA) Soluble Protein Flex Set and equal volumes of the samples were pooled. The CBA assay was performed on culture supernatants without any dilution. These details have been included in the revised manuscript. Live cell imaging: It appears that information in methods contradicts the data shown in Figure 2. According to the figure and supplied raw data, imaging was performed over a course of over 76 hours, while methods section states “48 hours”. Live cell imaging: The imaging was performed till 76 hours and it was a mistake to mention this as 48 hours in the method section, we thank you for pointing out this anomaly and this has been corrected in the revised manuscript. Raw data: Data for Figure 1: it might be easier to understand the data if columns were labeled by cytokine and sample (e.g. IL8 negative selection, IL8 positive selection, etc. as opposed to IL8B, IL8C, etc.) Suggested changes have been made. Data for Figure 2: were 36 fields for each sample for each time point individually counted, as the information on the figure indicates? Raw data appears to have only the averages. Was the distribution of counts tight, or was there high variation? Including raw data for individual field counts might be more appropriate . During the live cell imaging experiment images were logged and later cells were counted using the Cell-IQ Analyser software. The protocol for cell counting in the analysis software was assembled to finally provide the average number of cell count in the selected fields. The cell counts from individual fields of various images were embedded in the analysis software, thought it was possible to export these tables but due to very large number of data files we focussed only on the averages. Discussion: Another option to positively isolate monocytes is to use anti-CD33 instead of anti-CD14 coated beads. This may be more appropriate in studies that aim to measure response to LPS, when positive selection is preferred (e.g. in cases of limited sample from which sequential separation of different lymphocyte subsets is desired). It might be worthy to discuss this option. This is a good suggestion keeping in mind that the CD14 is an endogenous ligand for CD33 in monocyte-derived immature dendritic cells. It has been reported (Hiroshi Nakada et al.; J Biol Chem. 2014 Sep 5; 289(36): 25341–25350) that when monocyte-derived immature dendritic cells were stimulated with LPS in the presence of anti-CD33Ab, the production of IL-12 and phosphorylation of NF-κB decreased significantly. Most likely the positively isolated monocytes using CD33-Ab beads would behave in a similar manner. This possibility has been discussed in the Discussion section of the revised manuscript. Citations: Several citations throughout the paper appear to be inaccurate; for example, in Introduction some citations on altered behavior of cells following microbead attachment: We once again thank the reviewer for thoroughly examining the citations; the list of references has been appropriately revised. The differences between the findings of Beliakova-Bethell et al. and Neu et al. & Elkord et al. have been incorporated in the revised manuscript. Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 22 Dec 2017 Pramod Upadhyay , National Institute of Immunology, New Delhi, India 22 Dec 2017 Author Response Thank you for providing us valuable comments. We have addressed all of them in the revised version of the manuscript. Specifically Methods: Cytometic bead array: there are several ... Continue reading Thank you for providing us valuable comments. We have addressed all of them in the revised version of the manuscript. Specifically Methods: Cytometic bead array: there are several cytometric bead arrays available for human samples (e.g. “human inflammatory cytokine kit”, “human chemokine kit”). Which one was used? Legend for Figure 1 indicates that 5 samples were pooled. It would be helpful to provide details on how the pooling was done (the entire samples, equal volumes, equal proportions?) and what dilutions were made for the assay. We used Cytometric Bead Array (CBA) Soluble Protein Flex Set and equal volumes of the samples were pooled. The CBA assay was performed on culture supernatants without any dilution. These details have been included in the revised manuscript. Live cell imaging: It appears that information in methods contradicts the data shown in Figure 2. According to the figure and supplied raw data, imaging was performed over a course of over 76 hours, while methods section states “48 hours”. Live cell imaging: The imaging was performed till 76 hours and it was a mistake to mention this as 48 hours in the method section, we thank you for pointing out this anomaly and this has been corrected in the revised manuscript. Raw data: Data for Figure 1: it might be easier to understand the data if columns were labeled by cytokine and sample (e.g. IL8 negative selection, IL8 positive selection, etc. as opposed to IL8B, IL8C, etc.) Suggested changes have been made. Data for Figure 2: were 36 fields for each sample for each time point individually counted, as the information on the figure indicates? Raw data appears to have only the averages. Was the distribution of counts tight, or was there high variation? Including raw data for individual field counts might be more appropriate . During the live cell imaging experiment images were logged and later cells were counted using the Cell-IQ Analyser software. The protocol for cell counting in the analysis software was assembled to finally provide the average number of cell count in the selected fields. The cell counts from individual fields of various images were embedded in the analysis software, thought it was possible to export these tables but due to very large number of data files we focussed only on the averages. Discussion: Another option to positively isolate monocytes is to use anti-CD33 instead of anti-CD14 coated beads. This may be more appropriate in studies that aim to measure response to LPS, when positive selection is preferred (e.g. in cases of limited sample from which sequential separation of different lymphocyte subsets is desired). It might be worthy to discuss this option. This is a good suggestion keeping in mind that the CD14 is an endogenous ligand for CD33 in monocyte-derived immature dendritic cells. It has been reported (Hiroshi Nakada et al.; J Biol Chem. 2014 Sep 5; 289(36): 25341–25350) that when monocyte-derived immature dendritic cells were stimulated with LPS in the presence of anti-CD33Ab, the production of IL-12 and phosphorylation of NF-κB decreased significantly. Most likely the positively isolated monocytes using CD33-Ab beads would behave in a similar manner. This possibility has been discussed in the Discussion section of the revised manuscript. Citations: Several citations throughout the paper appear to be inaccurate; for example, in Introduction some citations on altered behavior of cells following microbead attachment: We once again thank the reviewer for thoroughly examining the citations; the list of references has been appropriately revised. The differences between the findings of Beliakova-Bethell et al. and Neu et al. & Elkord et al. have been incorporated in the revised manuscript. Thank you for providing us valuable comments. We have addressed all of them in the revised version of the manuscript. Specifically Methods: Cytometic bead array: there are several cytometric bead arrays available for human samples (e.g. “human inflammatory cytokine kit”, “human chemokine kit”). Which one was used? Legend for Figure 1 indicates that 5 samples were pooled. It would be helpful to provide details on how the pooling was done (the entire samples, equal volumes, equal proportions?) and what dilutions were made for the assay. We used Cytometric Bead Array (CBA) Soluble Protein Flex Set and equal volumes of the samples were pooled. The CBA assay was performed on culture supernatants without any dilution. These details have been included in the revised manuscript. Live cell imaging: It appears that information in methods contradicts the data shown in Figure 2. According to the figure and supplied raw data, imaging was performed over a course of over 76 hours, while methods section states “48 hours”. Live cell imaging: The imaging was performed till 76 hours and it was a mistake to mention this as 48 hours in the method section, we thank you for pointing out this anomaly and this has been corrected in the revised manuscript. Raw data: Data for Figure 1: it might be easier to understand the data if columns were labeled by cytokine and sample (e.g. IL8 negative selection, IL8 positive selection, etc. as opposed to IL8B, IL8C, etc.) Suggested changes have been made. Data for Figure 2: were 36 fields for each sample for each time point individually counted, as the information on the figure indicates? Raw data appears to have only the averages. Was the distribution of counts tight, or was there high variation? Including raw data for individual field counts might be more appropriate . During the live cell imaging experiment images were logged and later cells were counted using the Cell-IQ Analyser software. The protocol for cell counting in the analysis software was assembled to finally provide the average number of cell count in the selected fields. The cell counts from individual fields of various images were embedded in the analysis software, thought it was possible to export these tables but due to very large number of data files we focussed only on the averages. Discussion: Another option to positively isolate monocytes is to use anti-CD33 instead of anti-CD14 coated beads. This may be more appropriate in studies that aim to measure response to LPS, when positive selection is preferred (e.g. in cases of limited sample from which sequential separation of different lymphocyte subsets is desired). It might be worthy to discuss this option. This is a good suggestion keeping in mind that the CD14 is an endogenous ligand for CD33 in monocyte-derived immature dendritic cells. It has been reported (Hiroshi Nakada et al.; J Biol Chem. 2014 Sep 5; 289(36): 25341–25350) that when monocyte-derived immature dendritic cells were stimulated with LPS in the presence of anti-CD33Ab, the production of IL-12 and phosphorylation of NF-κB decreased significantly. Most likely the positively isolated monocytes using CD33-Ab beads would behave in a similar manner. This possibility has been discussed in the Discussion section of the revised manuscript. Citations: Several citations throughout the paper appear to be inaccurate; for example, in Introduction some citations on altered behavior of cells following microbead attachment: We once again thank the reviewer for thoroughly examining the citations; the list of references has been appropriately revised. The differences between the findings of Beliakova-Bethell et al. and Neu et al. & Elkord et al. have been incorporated in the revised manuscript. Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 3 VERSION 3 PUBLISHED 23 Nov 2017 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 Version 3 (revision) 28 Mar 18 Version 2 (revision) 22 Dec 17 read Version 1 23 Nov 17 read Nadejda Beliakova-Bethell , VA San Diego Healthcare System, San Diego, USA Aniruddha Roy , Birla Institute of Technology & Science, Pilani (BITS), Pilani, India Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2018 Roy A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 23 Mar 2018 | for Version 2 Aniruddha Roy , Department of Pharmacy, Birla Institute of Technology & Science, Pilani (BITS), Pilani, India 0 Views copyright © 2018 Roy A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions In the current manuscript, the authors have investigated the impact of positive selection and negative selection on the activation, function, and proliferation of monocytes. It is an important study as many researchers use these techniques interchangeably, greatly impacting the final outcome of the study and erroneous conclusion. Though the current study has used only a few experiments to draw the conclusion, their study design was neat. The authors may clarify some minor points to make the manuscript better: In the introduction section, the authors have written: "CD14 lacks a cytoplasmatic domain and such cells are reported to function in a restricted manner" - a brief explanation of what is meant by restricted manner would be helpful. The sentence on page number 5 reads: 'These were the cells from which the microbeads were either degraded or " detected " and cells were without microbeads.' : Probably instead of detected, it will be detached. Is the size of the beads known? Can there be a size-dependent effect due to steric hindrance of the CD14 bound beads to the TLR4? If only antibody is used, can we see the same effect? The main function of CD14 in LPS signaling in monocytes is it binds with LPS binding protein (LBP). The role of LBP appears to be that of aiding LPS to dock at the LPS receptor complex by initially binding LPS and then forming a ternary complex with CD14, thus enabling LPS to be transferred to the LPS receptor complex composed of TLR4 and MD-2. As in these in vitro studies, LBP has not been used, probably CD14 may not directly affect the LPS binding with the TLR4 receptor. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (1) Author Response 28 Mar 2018 Pramod Upadhyay, National Institute of Immunology, New Delhi, India Thank you for providing us valuable comments. We have addressed all of them in the revised version of the manuscript. Specifically In the introduction section, the authors have written: "CD14 lacks a cytoplasmatic domain and such cells are reported to function in a restricted manner" - a brief explanation of what is meant by restricted manner would be helpful. It was an inadvertent mistake to write an incomplete sentence. This has been corrected in the revised manuscript. The sentence on page number 5 reads: 'These were the cells from which the microbeads were either degraded or "detected" and cells were without microbeads.' : Probably instead of detected, it will be detached. The correction has been made in the revised manuscript. Is the size of the beads known? Can there be a size-dependent effect due to steric hindrance of the CD14 bound beads to the TLR4? If only antibody is used, can we see the same effect? The main function of CD14 in LPS signaling in monocytes is it binds with LPS binding protein (LBP). The role of LBP appears to be that of aiding LPS to dock at the LPS receptor complex by initially binding LPS and then forming a ternary complex with CD14, thus enabling LPS to be transferred to the LPS receptor complex composed of TLR4 and MD-2. As in these in vitro studies, LBP has not been used, probably CD14 may not directly affect the LPS binding with the TLR4 receptor. Nano meter size beads are used is cells isolation and these are unlikely to create significant steric hindrance. The main point is that once Ab-conjugated beads get bound to the antigen cell surface (CD14 in present case); it is bound to influence the expression, transcription and translation machinery inside the cell. And secondly, as a result of conjugation of cell surface CD14 molecule with Ab-conjugated bead in cells isolation step, the cell surface CD14 molecule would no longer be available for LBP to initiate the activation cascade. This has been elaborated in the Introduction section of the revised manuscript. View more View less Competing Interests There are competing interests. reply Respond Report a concern Roy A. Peer Review Report For: Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour [version 3; peer review: 2 approved] . F1000Research 2018, 6 :2045 ( https://doi.org/10.5256/f1000research.14717.r31885) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/6-2045/v2#referee-response-31885 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2017 Beliakova-Bethell N. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 12 Dec 2017 | for Version 1 Nadejda Beliakova-Bethell , VA San Diego Healthcare System, San Diego, CA, USA 0 Views copyright © 2017 Beliakova-Bethell N. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions This study assesses monocyte function following isolation of these cells from total peripheral blood mononuclear cells (PBMC) using positive or negative immunomagnetic selection. It addresses an important topic, because cell isolation procedure may affect cell function and skew conclusions obtained from in vitro studies of immune cells isolated from blood. Despite a limited assessment of functions and isolation methods, this study should have an impact on selection of monocyte isolation protocol when these cells are intended for investigation of specific responses, which this study tested. The study design is appropriate and conclusions drawn are adequately supported by the results. The paper may be improved, however, by providing more details in methods, raw data and discussion, and by careful revision of citations as detailed below: Methods: Cytometic bead array: there are several cytometric bead arrays available for human samples (e.g. “human inflammatory cytokine kit”, “human chemokine kit”). Which one was used? Legend for Figure 1 indicates that 5 samples were pooled. It would be helpful to provide details on how the pooling was done (the entire samples, equal volumes, equal proportions?) and what dilutions were made for the assay. Live cell imaging: It appears that information in methods contradicts the data shown in Figure 2. According to the figure and supplied raw data, imaging was performed over a course of over 76 hours, while methods section states “48 hours”. Raw data: Data for Figure 1: it might be easier to understand the data if columns were labeled by cytokine and sample (e.g. IL8 negative selection, IL8 positive selection, etc. as opposed to IL8B, IL8C, etc.) Data for Figure 2: were 36 fields for each sample for each time point individually counted, as the information on the figure indicates? Raw data appears to have only the averages. Was the distribution of counts tight, or was there high variation? Including raw data for individual field counts might be more appropriate. Discussion: Another option to positively isolate monocytes is to use anti-CD33 instead of anti-CD14 coated beads. This may be more appropriate in studies that aim to measure response to LPS, when positive selection is preferred (e.g. in cases of limited sample from which sequential separation of different lymphocyte subsets is desired). It might be worthy to discuss this option. Citations: Several citations throughout the paper appear to be inaccurate; for example, in Introduction some citations on altered behavior of cells following microbead attachment: (1) Safarik and Safarikova is a method review, and Horgan et al. is a protocol; neither studies cell behavior; (2) Semple et al. observed no functional changes in limited tests they did; the difference that they report pertains to CD19+ vs CD19- cells, not positively isolated vs negatively isolated cells; (3) Fuchslin et al. labeled bacterial cells for their quantification in water; and Ribaut et al. labeled parasites for their isolation from infected blood for research purposes; the relevance of these two citations for the present study may be questionable. In Discussion, Adams et al. describe a special case of MACS for multitarget sort, and may not be appropriate for referencing commonality of magnetic sorting. While the results from the present study are consistent with gene expression studies by Beliakova-Bethell et al. in terms of effects of positive selection, the reference to Beliakova-Bethell et al. is made following description of Neu et al. and Elkord et al. studies, stating that the findings were similar. This is not accurate because Neu et al. and Elkord et al. performed functional assessment following cell separation, while Beliakova-Bethell et al. lysed the cells immediately after isolation and did not measure cell function. Instead of the statement of similarity of findings, it might be worth pointing out that positive selection affects monocytes the most both in the short term and in the long term. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Virology, immunology, cell separation and flow cytometry, analysis of gene expression I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (1) Author Response 22 Dec 2017 Pramod Upadhyay, National Institute of Immunology, New Delhi, India Thank you for providing us valuable comments. We have addressed all of them in the revised version of the manuscript. Specifically Methods: Cytometic bead array: there are several cytometric bead arrays available for human samples (e.g. “human inflammatory cytokine kit”, “human chemokine kit”). Which one was used? Legend for Figure 1 indicates that 5 samples were pooled. It would be helpful to provide details on how the pooling was done (the entire samples, equal volumes, equal proportions?) and what dilutions were made for the assay. We used Cytometric Bead Array (CBA) Soluble Protein Flex Set and equal volumes of the samples were pooled. The CBA assay was performed on culture supernatants without any dilution. These details have been included in the revised manuscript. Live cell imaging: It appears that information in methods contradicts the data shown in Figure 2. According to the figure and supplied raw data, imaging was performed over a course of over 76 hours, while methods section states “48 hours”. Live cell imaging: The imaging was performed till 76 hours and it was a mistake to mention this as 48 hours in the method section, we thank you for pointing out this anomaly and this has been corrected in the revised manuscript. Raw data: Data for Figure 1: it might be easier to understand the data if columns were labeled by cytokine and sample (e.g. IL8 negative selection, IL8 positive selection, etc. as opposed to IL8B, IL8C, etc.) Suggested changes have been made. Data for Figure 2: were 36 fields for each sample for each time point individually counted, as the information on the figure indicates? Raw data appears to have only the averages. Was the distribution of counts tight, or was there high variation? Including raw data for individual field counts might be more appropriate . During the live cell imaging experiment images were logged and later cells were counted using the Cell-IQ Analyser software. The protocol for cell counting in the analysis software was assembled to finally provide the average number of cell count in the selected fields. The cell counts from individual fields of various images were embedded in the analysis software, thought it was possible to export these tables but due to very large number of data files we focussed only on the averages. Discussion: Another option to positively isolate monocytes is to use anti-CD33 instead of anti-CD14 coated beads. This may be more appropriate in studies that aim to measure response to LPS, when positive selection is preferred (e.g. in cases of limited sample from which sequential separation of different lymphocyte subsets is desired). It might be worthy to discuss this option. This is a good suggestion keeping in mind that the CD14 is an endogenous ligand for CD33 in monocyte-derived immature dendritic cells. It has been reported (Hiroshi Nakada et al.; J Biol Chem. 2014 Sep 5; 289(36): 25341–25350) that when monocyte-derived immature dendritic cells were stimulated with LPS in the presence of anti-CD33Ab, the production of IL-12 and phosphorylation of NF-κB decreased significantly. Most likely the positively isolated monocytes using CD33-Ab beads would behave in a similar manner. This possibility has been discussed in the Discussion section of the revised manuscript. Citations: Several citations throughout the paper appear to be inaccurate; for example, in Introduction some citations on altered behavior of cells following microbead attachment: We once again thank the reviewer for thoroughly examining the citations; the list of references has been appropriately revised. The differences between the findings of Beliakova-Bethell et al. and Neu et al. & Elkord et al. have been incorporated in the revised manuscript. View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Beliakova-Bethell N. Peer Review Report For: Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour [version 3; peer review: 2 approved] . F1000Research 2018, 6 :2045 ( https://doi.org/10.5256/f1000research.13872.r28324) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/6-2045/v1#referee-response-28324 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions Click here to access the data. The problem Spreadsheet data files may not format correctly if your computer is using different default delimiters (symbols used to separate values into separate cells) - a spreadsheet created in one region is sometimes misinterpreted by computers in other regions. You can change the regional settings on your computer so that the spreadsheet can be interpreted correctly. How to fix it Save downloaded CSV file Open spreadsheet program (e.g. Excel) Click the ‘Data’ tab at the top Click the ‘From text’ icon (top left) Browse for downloaded CSV file, click ‘Import’ Ensure ‘Delimited’ radio button is selected, click ‘Next’ Check one of the appropriate delimiter checkboxes (you can visualize the formatting by looking at the data preview below these options) Click ‘Finish’ Downloaded data do not display as expected? Download the data (0.31KB) Dataset citation: Bhattacharjee J, Das B, Mishra A et al. . Dataset 1 in: Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour. F1000Research 2018, 6 :2045 (https://doi.org/10.5256/f1000research.12802.d188477) Close Click here to access the data. The problem Spreadsheet data files may not format correctly if your computer is using different default delimiters (symbols used to separate values into separate cells) - a spreadsheet created in one region is sometimes misinterpreted by computers in other regions. You can change the regional settings on your computer so that the spreadsheet can be interpreted correctly. How to fix it Save downloaded CSV file Open spreadsheet program (e.g. Excel) Click the ‘Data’ tab at the top Click the ‘From text’ icon (top left) Browse for downloaded CSV file, click ‘Import’ Ensure ‘Delimited’ radio button is selected, click ‘Next’ Check one of the appropriate delimiter checkboxes (you can visualize the formatting by looking at the data preview below these options) Click ‘Finish’ Downloaded data do not display as expected? Download the data (6.83KB) Dataset citation: Bhattacharjee J, Das B, Mishra A et al. . Dataset 2 in: Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour. F1000Research 2018, 6 :2045 (https://doi.org/10.5256/f1000research.12802.d182357) Close Click here to access the data. The problem Spreadsheet data files may not format correctly if your computer is using different default delimiters (symbols used to separate values into separate cells) - a spreadsheet created in one region is sometimes misinterpreted by computers in other regions. You can change the regional settings on your computer so that the spreadsheet can be interpreted correctly. How to fix it Save downloaded CSV file Open spreadsheet program (e.g. Excel) Click the ‘Data’ tab at the top Click the ‘From text’ icon (top left) Browse for downloaded CSV file, click ‘Import’ Ensure ‘Delimited’ radio button is selected, click ‘Next’ Check one of the appropriate delimiter checkboxes (you can visualize the formatting by looking at the data preview below these options) Click ‘Finish’ Downloaded data do not display as expected? Download the data (0.10KB) Dataset citation: Bhattacharjee J, Das B, Mishra A et al. . Dataset 3 in: Monocytes isolated by positive and negative magnetic sorting techniques show different molecular characteristics and immunophenotypic behaviour. F1000Research 2018, 6 :2045 (https://doi.org/10.5256/f1000research.12802.d182358) Close Adjust parameters to alter display View on desktop for interactive features Includes Interactive Elements View on desktop for interactive features Competing Interests Policy Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. Consider the following examples, but note that this is not an exhaustive list: Examples of 'Non-Financial Competing Interests' Within the past 4 years, you have held joint grants, published or collaborated with any of the authors of the selected paper. You have a close personal relationship (e.g. parent, spouse, sibling, or domestic partner) with any of the authors. 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Register $(document).ready(function () { signIn.createSignInAsRow($("#sign-in-form-gfb-popup")); $(".target-field").each(function () { var uris = $(this).val().split("/"); if (uris.pop() === "login") { $(this).val(uris.toString().replace(",","/")); } }); });