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Mitomycin C treatment to prepare feeder cells

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#1 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,


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