Hello everyone I need help,
I am working with KGN granulosa cell line (DMEM/F12 media, 10% FBS, 1% Pen/Strep)
I am treating the cells with PMA at 100nM concentration to activate PKC, but everytime I add PMA my cells are shrinking and look like they are dying.. Does anyone have ever experienced something similar ? Any idea why they are looking like that and why does the PMA would induce that ? (I also tried with lower concentrations; 20. and 40nm and had the same results..).
Thank you so much
Hello Fortin,
If you have the chance I would suggest checking the water levels and the CO2 infusion of your incubator, I have noticed that even though sometimes not apparent slide changes on these basic conditions can hit hard your culturing.
Alternatively, you can slowly increase the % of FBS and see if they can come back to how they are supposed to look like.
Hope it helps,
Marcos
Thank you very much, I’ve checked and CO2 have been constant at 5%, water is ok too! I’ve noticed that after 16-24h they start looking normal again.. but I’m still worried because ultimately I will treat them for only 12h and so the results will be skewed by this problem..
Chloe
PMA can induce cell cycle arrest, apoptosis or non-apoptotic cell death in some cell lines. The effect depends on cell context. For instance, in LNCaP cells, treatment with PMA induces PKC-dependent apoptosis through activation of JNK, but in AsPC1 cells this treatment induces non-apoptotic and PKC-independent cell death.
Chloe, Your cells are not shrinking, they are being stimulated!
As Roman identifies, PMA is a mitogen that stimulates PKC. What does PKC control? Cell cycle and proliferation. Your cells are obviously adherent, but when they are preparing to divide they must round up and appear to get smaller. Their volume isn't changing, merely their 3 dimensional shape. As they divide they detach from the surface to allow the two daughter cells to separate. If you shake your flask when they are in this phase, many will detach and float, looking just like dead cells. Allow them to settle and they will reattach and soon look 'normal' again, with many fewer 'dead' cells!
What you are observing is normal. Your cells are behaving just as I would expect them to!
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