Isolation of murine large intestinal crypt cell populations with flow sorting

Abstract Here we present a high-resolution sorting protocol for colon crypt stem cells, their daughter cells and mature, differentiated cell types. We used freshly dissected mouse colons and validated intestinal cell surface markers amenable to Flow Activated Cell Sorting (FACS). This 5-7 hour protocol enables isolation of six distinct cryptal cell populations (Stem, AbsPro, SecPDG, Tuft, Ent, and EEC) from any mouse strain/background (Figure 1 and 2) . Downstream analysis of sorted cells (Transcriptomics = bulk RNA-seq and Proteomics = small cell number LC/MS) validated the identity of cell populations. An important strength of this protocol is the independence from any trans-genic labeling of cell types and the flexibility for users to add additional markers for a variety of downstream applications. The absence of proteases during dissociation increases antigen expression resolving the six cell types but also decreases cell yield (Figure 3). The main steps of this protocol include: Tissue Dissection, Tissue Dissociation, Preparation for FACS, and Performing FACS.