I model the population dynamics of several (adherent) cell lines. To that aim I want to characterize the growth dynamics of those lines experimentally. We already measured the growthrate (=birth - death rate) in a simple proliferation assay based on cell counting. It would be very helpful to have separate information on either birth or death rate to incorporate the parameters to the theoretical model.
We tried to measure the basal apoptosis rate by Annexin V/PI staining in FACS. Unfortunately, the populations were not clearly distiguishable, probably because the fraction of apoptotic and dead cells in the population is very small (since I am interested in the basal rate, the experiment was perfomed without treatment).
Does anybody have some experience or ideas on how to measure the basal apoptosis rate or the fraction of apoptotic and dead cells in a cell population?
Thanks a lot in advance.
Hi Nina,
Replying to your request on measuring the basal apoptosis rate or the fraction of apoptotic and dead cells in a cell population, two points have to be taken into consideration, one of general cell culture nature of apoptosis, and another related cancer cell lines. Generally, it was found that measuring the number of apoptotic cells before plating, say, the PBMC did not reflect the extent of PBMC injury, but measuring apoptotic cell frequencies at the end of the assay DID. In other words, post- rather than pre-T cell assay detection of apoptotic cells in PBMC indicates impaired T cell function. To this end, bystander apoptotic cells do not exert a paracrine effect on T cell activation. As for cancer cells, it was also shown that early signaling in camptothecin-treated MCF-7 cells followed an intrinsic pathway, but death was delayed and late events exhibited few hallmarks of apoptosis. Noteworthy, cell death induced by CPT in the MCF-7 breast cancer cell line is largely caspase-independent. Thus CPT treatment activated both an apoptotic response as well as an adaptive, autophagic response in normal breast epithelial cells and non-invasive MCF-7 cells, which died an accelerated death when autophagy was inhibited during CPT exposure. On the other hand, the metastatic breast cancer cell lines BT-549, MDA-MB-231 and MDA-MB-435, all were resistant to concentrations of CPT that killed, say, HMEC, MCF-7 and T47D lines, either in the presence or absence of autophagy inhibitors. The inability of invasive breast cancer cell lines to activate apoptosis in response to both DNA damage and autophagy inhibition suggests that the autophagic survival response may either be independent of apoptosis or irrelevant in cells that have acquired resistance to genotoxic stress stimuli. The DELAYED APOPTOSIS in the noninvasive tumor cells could stem from a defective apoptotic pathway, contributed to by aberrant expression of an intermediate such as caspase-3, further aided by a 'defective' autophagic pathway
Best wishes,
Ilya
Hi Nina,
Why do you expect to see apoptotic cells in untreated cancer cell line culture? If they growth in complete media at an appropriate concentration, where should not be any apoptosis. After they reach >80% of confluency with no media change, 7-AAD/Annexin V staining gives 0,1-1% of apoptotic cells for the most of the cancer cell lines. To detect higher apoptotic rate you need to treat them.
If you want to characterize the growth dynamics, I would suggest to use CFSE assay.
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