Hello, everyone. Mi name is Jorge, and I'm student in Universidad de Guanajuato in Mexico. The last weeks, I have cultured primary leukocytes in DMED with 1% of penicillin-streptomycin, and 20ug/ml of Concanavalin A; but my cells don't increase in number in 24 hours, 48 hours, or 36 hours.
I obtained the cells with Ficoll-hypaque PLUS from healthy patients, and I used P60 dishes.
I hope can you help me! :)
Hello Jorge Luis Tejeda Salazar
You should culture leukocytes in RPMI media containing 10% FBS instead of DMEM as leukocytes will grow well in RPMI media. Leukocytes do not readily proliferate without stimulus. To induce proliferation, mitogens such as phytohemagglutinin (PHA) or lipopolysaccharide (LPS) may be added to the culture medium at a concentration of 1-5 µg/mL.
I use PHA which is widely used for the purpose of mitotic stimulation of human lymphocytes. Additionally, you may add IL-2 at a final concentration of 100 IU/ml. The cells express high affinity receptor for IL-2 and proliferate in response to this cytokine.
Best.
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