T cell proliferation - problems (Sep/19/2003 )

I'm looking at human PBMC response to various protein antigens. Quite straightforward if you think about it. Cells are incubated at 1 million/ml or 200,000/well in 96 well plate in RPMI 1640 + 10% AB sera + 2mM L- glutamine. Reaction allowed to go for 6 days before pulsing for 18 hrs with uCi. Incubated with 5% carbon dioxide and 37 degrees. So far however, I have been getting really low counts onr eading the plates. only ~ 1000 for control of cells only when I would expect closer to 10,000 and even for my positive control ConA, only counts of 10-20,000 when I should see something closer to 100,00. I've tried pulsing at 4 days but no luck. any ideas whats wrong?

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Dear miclino,

Normally the peak of proliferation for mitogens (Con A, PHA, etc.) is about 2-3 days and for antigens is 4-6 days. It's means that you should take your cultures to harverster at these times... If you left your cultures with ConA with 6 days of incubation, probably all cells was already died.

Doesn't the matter if you incubate with thymidine for 6 hours, 18 hours or even days. The results will be pretty the same...

Try to see the concentration of the tritiated-thymidine that you are using. I use at 1 uCi per well diluted in culture medium (pulsed for 6 hours).

About your controls being so low, I think could be the long time of culture (6 days + 18 hs). Try to reduce your time of incubation.

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