I need to evaluate the % of T cells, neutrophils and dendritic cells in skin sample by flow cytomtery. I would really appreciate any suggestions for making effective cell suspension from the skin. Thanking you in advance.
You can use gentle MACS instrument from MACS for preparing single cell suspension. You can also use tea-strainer or cell strainer (BD Pharmingen) for making single cell suspension. for tea strainer, put the skin portion between two pieces of wire-mesh and pour 2-3 ml of cell culture media. crush the tissue by using blunt ended thing like scissor etc. than collect all the cells in 15 ml centrifuge tube and spin at 4 degree centigrade for 6 mins at 1800 rpm. Dissolve the pellet in 1ml media than add around 4-5 ml of RBC lysis solution(155mM NH4 Cl; 10mM KHCO3; 0,1mM EDTA; pH 7.3)/Gray RBC lysis buffer. keep at room temp. for 10-12 mins, when solution loses its red colour, than centrifuge as earlier n resuspend the pellet in 1 ml cell culture media and count the live cells (Trypan blue etc.) now cell is ready for culture. all d best
Thank you Sir. I would be grateful if you provide any enzymatic methods.
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