Problem with mitogen stimulation?

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RockyDoc
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Problem with mitogen 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! Based on the literature, I was expecting the cells to grow well in the presence of the mitogens. Any ideas PLEASE?

samm
samm's picture
Have you seen differences

Have you seen differences based on amounts of ConA or Ionomycin?

Working with primary mouse T cells, I'd found there is a "sweet spot" of max proliferation/min cell death & consequently max cell numbers depending upon the strength of primary signal (here the mitogeens ConA or PMA+I) and the costimulatory profile thereby engaged. Too strong a signal may lead to a)AICD and b) predominantly inhibitory costimulatory profile for CTLA4.

Also, how are you measuring viabilty? MTT? PI-Via? Have you done a PI-cell cycle analysis?

RockyDoc
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I used various combinations

I used various combinations of Con A (3) and PMA/IoM (8). There was no difference between any of the treatments, just between mitogen-samples and controls. I am using low, medium and high concentrations of mitogens - all of which are reported in the literature for stimulation in the Jurkats.

I am using the FDA/EtBr viability assay and cell counts as preliminary tools before going onto the MTT assay. This is why I noticed the 10-fold difference in cell number at t=96h. The assays will be repeated but I will also move onto the mTT to see how that goes.

Do you think I should look at closer time points such as 6h and 12 h? However, work on Jurkats in the lit has looked the mitogen stimulation up to 96 h. Frustrating!

Thanks for your help on this!

samm
samm's picture
Try working it out with PMA+I

Try working it out with PMA+I first - should be easier. PMA 1-100ng/ml range (1, 5, 10, 20, 50, 100) and ionomycin (0.05-5 uM).
Also, how many cells are you using? If its a 96 well plate, 10k is enough for either EtBr/PI-via cell counts - with perhaps 3 wells req for the PI-cellcyc on a regular flow cytometer, or even 1 on a Guava.
Time points of 6 and 12 h are too early for cell cycle assays (12 can be used as a time point, but I've usually seen differences at 24 and 48 h).

I think you are somehow killing off your cells in the mitogen treated conditions - which can be rectified if you can attain a balance between cell number and mitogen concentration. Also ensure that you don't have any contaminants (bugs - mycoplasma) etc associated with the cells - unlikely for Jurkats, but possible.

RockyDoc
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Hi Samm,

Hi Samm,

The concentrations that I have been using are up to 100ng/ml PMA and up to 1uM IoM. My cells are not contaminated and I am seeding at a density of 1 x 10(5) per ml (2 x 10(4) per well). Basically I am replicating what has been reported in the literature.

I plan to repeat the experiment again this week to see how things turn out. Will keep you posted and thanks again for your time and help.

samm
samm's picture
Try reducing your Iono

Try reducing your Iono concentration to 0.25 uM, and titre PMA across the range. I did see that high Ionomycin (>1 uM) just kills off the cells, high PMA saturates without killing.
All the best!