Here's another suggestion...hope it works! I have found many times
that certain cell lines look fine and healthy upon thawing, but then
crash by the next day. To help the cells from the start I thaw them
onto irradiated feeder cells (mouse thymocytes, spenocytes--3000RADS)
at a density of 10^5 feeder cells per well in a 96 well plate. Normally
I have my frozen cells at 5 x 10^6 cells per vial. I bring them up in
10ml of media and add 100 microliters per well. Within 2 weeks (maybe
4 in some cases) you should see viable cells growing out. The feeder
layer will die off and you can continue to expand them up to a 24 well
plate until they get going. I would suggest keeping the cells dense
until you know they are heathy (>90% viable). I'm not sure why you
have continued growing the cells with HT present...once the cells have
been selected (HAT, then HT for maybe 2 passages) the HT is no longer
needed. Good luck! This may seem like a lot of work but believe me
it has been a savior for me!
_______________________________________
Dave Plas
PLAS_D at WUMS.WUSTL.EDU
Washington University
