Cell Population Analyses During Skin Carcinogenesis

Abstract

Cancer development is a multiple-step process involving many cell types including cancer precursor cells, immune cells, fibroblasts and endothelial cells. Each type of cells undergoes signaling and functional changes during carcinogenesis. The current challenge for many cancer researchers is to dissect these changes in each cell type during the multiple-step process in vivo. In the last few years, the authors have developed a set of procedures to isolate different cell populations during skin cancer development using K14creER/R26-SmoM2 YFP mice.The procedure is divided into 6 parts: 1) generating appropriate mice for the study (K14creER + and R26-SmoM2 YFP+ mice in this protocol); 2)inducing SmoM2 YFP expression in mouse skin; 3) preparing mouse skin biopsies; 4) isolating epidermis from skin; 5) preparing single cells from epidermis; 6) labeling single cell populations for flow cytometry analysis. Generation of sufficient number of mice with the right genotype is the limiting step in this protocol, which may take up to two months. The rest of steps take a few hours to a few days. Within this protocol, we also include a section for troubleshooting. Although we focus on skin cancer, this protocol may be modified to apply for other animal models of human diseases.

Isolating and Cryo-Preserving Pig Skin Cells for Single Cell RNA Sequencing Study

“…A dissociation method with high cell yield and cell viability can minimize the volume of skin sample, and allow detecting cell types with small numbers. To date, a few methods and protocols have been published for preparing skin single cells of rodents [51][52][53][54][55][56][57] and human [58][59][60][61][62][63] via enzymatic digestion and mechanical dissociation. These methods diverge in the enzyme types, concentrations, exposure time, temperature 51,52,[61][62][63][53][54][55][56][57][58][59][60] .…”

“…To date, a few methods and protocols have been published for preparing skin single cells of rodents [51][52][53][54][55][56][57] and human [58][59][60][61][62][63] via enzymatic digestion and mechanical dissociation. These methods diverge in the enzyme types, concentrations, exposure time, temperature 51,52,[61][62][63][53][54][55][56][57][58][59][60] . Additionally, most of them use manual mechanical dissociation, such as dissection, mincing, agitation, pipetting or passing through syringes that are not efficient and consistent.…”