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Mitomycin C treatment to prepare feeder cells


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#1 borriello.87

borriello.87

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Posted 27 May 2011 - 03:30 PM

Hi all,
my boss told me to start T cell cloning experiments but I don't manage to obtain enough feeder cells.
Because we don't have an irradiator, I treat 10^7 PBMcs with 500ul RPMI+mitomycin C (final concentration 25ug/ml).
After incubating them at 37C 5%CO2 for 30', I wash at least 5 times with RPMI.
Finally, I count the cells and I find no more than 10^6 PBMCs!!
Could anyone give me some advice to improve the yield and tell me what's wrong with this protocol?
It would be wonderful to learn from your experience!!
Thanks in advance,

Francesco




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