Hi I am newbie on this topic, I am working with skin samples, we try to isolate basal keratinocyte, i found trypsinization with trypsin/EDTA 0,05% and 0,1% is not good enough, also I wonder how to isolate and purified basal keratinocyte ? i read people used percoll density gradient...we cant afford cell sorting assay or antibody marker..other people simply brush the sample or peeled of the epidermis? could that be done actually?
thanks
hello to purify keratinocytes the easiest way is to add geticinen (antibiotic== can not remember how to spell i but look it up in the literature) to remove fibroblasts and use fractional trypsinization i.e for short period to remove melanocytes (they come off really fast you have to watch under microscope) and when that is done you can just wash with PBS and then add your Keratinocyte full medium and depending on the amount of Calcium in your medium to keep them basal (low calcium around 0.2mM) or differentiated (high calcium (1.5mM).
I hope this helps
thanks souna, maybe you meant to say gentamicin? i have some using gentamicin+PBS for rinse solution does it really remove fibroblast? what do you mean with fractional trypsinization? i used the above trypsin solution though..but .i dont use gentamicyn
try finding the original paper to use gentamicin . when you use the keratinocyte medium it is usually enough to kill of most melaocytes. then use fractional trypsinization (i.e leave the trypsin on just enough to sea that the melanocyte retracted their extensions and started floating rinse with PBS add the complete keratinocyte medium and repeate the fractional trypsinization if you still see melanocytes
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