mitogenic stimulation

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RockyDoc
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mitogenic stimulation

I have been carrying out some preliminary work on Jurkat cells and the mitogens PMA/IoM and Concanavalin A. I incubed Jurkat cells with varying levels of PMA/IoM or Concanavalin A for 24 h, 48 h, 72 h and 96 h (today). The cell viability results have been coming out OK. All conditions are resulting in greater than 90% cell viability.

However, I have noticed that the cell numbers in the controls (cells only and carrier controls) are far greater than those in the test samples i.e. cell proliferation has been severely affected by the mitogens! I expected the cell to grow well in the presence of the mitogens. Any ideas PLEASE?

t136816
t136816's picture
Hi what concentration of

Hi what concentration of mitogens are you using? and what cell density are you plating your cells at?

RockyDoc
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Hi,

Hi,

I am still playing around with cell density and mitogen concentration.

RockyDoc

Tracy
Tracy's picture
RockyDoc wrote:Hi,

RockyDoc wrote:

Hi,

I am still playing around with cell density and mitogen concentration.

RockyDoc

Here is an example of the concentration of mitogen. Don't know if you used the similar.

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WM6-4CXMSNC-1&_coverDate=10%2F01%2F2004&_alid=511454853&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=6926&_sort=d&view=c&_acct=C000058478&_version=1&_urlVersion=0&_userid=2665235&md5=3d5c0bfcb6cb524b74fe781aa302aba0#SECX5

Mitogen stimulated lymphocyte proliferation and its inhibition by Curcumin
Mitogen stimulated lymphocyte proliferation was evaluated using a microculture assay [7]. Cells were plated in a 96-well plates at 0.2 million cells/well in 0.2 ml culture medium. Mitogen (Con A: 2 μg/ml or PHA: 5 μg/ml or PMA: 20 ng/ml) was added to the designated wells. Curcumin from 0 to 10 μg/ml was added to the designated wells and with equal distribution of carrier DMSO (0.1%) for vehicle control. Cells were cultured at 37°C in a humidified CO2 incubator with 5% CO2 in the air for 48 h. The cultures were pulsed with 3H-thymidine (ICN, 2 Ci/mmol) at 1 μCi/well for 16 h. Cells were harvested onto glass fibers (Skaton Filter Mat) using a Packard 96-well cell harvester. The incorporated radioactivity was determined with a Matrix-96 beta counter (Packard, Grove, IL).