Cells are stuck - (Nov/22/2005 )

Hi, I am growing keratinocytes and today I tried to passage them for the first time but the cells will not come off the flasks.

I tried trypsin/edtafor a few mins then removed it and put the flask in the incubator for another few mins. This did not work so I tried increasing the time I left the trypsin on for which again did not work. I then tried using 10x stronger trypsin edta no luck. So finally I incubated with trypsin edta for 10 mins then added PBS and centrifuged the solution and resuspended the pellet in media. I really don't think this has worked and I am at a loss over what I should do. Any advice?

hello again

those naughty keratinocytes

we use TE (has to be warmed to 37 to be effective). after aspirating the spnt, we add 2mL/10cm dish and let it sit at 37 for about 10-15min. too much more time is harmful to the cells, but less time won't pull them off the plate. after about 5 min, gently tap the plate on one side to dislodge them a bit, then put them back in...do that again at 10min... when they have uplifted (check with the scope) add 2 mL FCS +2mL KGM (or your favorite media) for recovery, spin at 1000rpm 5min, then dilute and seed. for experiments, we use PBS instead of the FCS/KGM and rinse the cells once with PBS then proceed with your protocol(for example, I would do it this way for RNA or DNA prepping from the cells)

if this doesn't work, your TE may be bad. try a fresh batch. repeated heating of TE makes it croak, so I would recommend small aliquots and heat it only once to 37. fresh TE works way better.

good luck

hello again

those naughty keratinocytes

we use TE (has to be warmed to 37 to be effective). after aspirating the spnt, we add 2mL/10cm dish and let it sit at 37 for about 10-15min. too much more time is harmful to the cells, but less time won't pull them off the plate. after about 5 min, gently tap the plate on one side to dislodge them a bit, then put them back in...do that again at 10min... when they have uplifted (check with the scope) add 2 mL FCS +2mL KGM (or your favorite media) for recovery, spin at 1000rpm 5min, then dilute and seed. for experiments, we use PBS instead of the FCS/KGM and rinse the cells once with PBS then proceed with your protocol(for example, I would do it this way for RNA or DNA prepping from the cells)

if this doesn't work, your TE may be bad. try a fresh batch. repeated heating of TE makes it croak, so I would recommend small aliquots and heat it only once to 37. fresh TE works way better.

good luck

Hi tried what you said but my cells are still stuck. I can't understand what I'm doing wrong.