Hello Guys,
I am trying to isolate keratinocytes from skin biopsies for single cell sequencing. I would like to have just keratinocytes without any immune cells or fibroblasts. Can anyone suggest a method? I am alreading inclined towards using dispase to separate the epidermis from the dermis, trypsin-EDTA to digest the epidermis and MACs to sort out CD45 positive cells. My worry is that my cells will not be viable afterwards.
the best method for isolation of human keratinocytes is two-step overnight digestion in 1.45-2.5 U/mL dispase followed by short (up to 3 minutes) cleavage in 0.25% trypsin with subsequent pipetting of the epidermis. Furthermore, it appears that the best results of culture of isolated primary keratinocytes may be achieved using collagen-coated culture vessels and serum-free EpiLife medium, which is also the most popular medium used for keratinocyte culture
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