The cell line I used in my experiment is a self-cultured porpoise fibroblast cell line. On the first day, I seeded cells on a 96-well plate and the cell density was 5 x 10^4 cells/mL. The day after, I added drugs and went for 24hr incubation. Then, on the third day, when I was going to perform the MTS assay, I found that all the cells were dead, including the cells of the control group which were only treated with a culture medium without any added drugs. I am so puzzled and wondering why, anyone has faced the same problem and can help me.
My cell culture medium is also without FBS since I need to collect the supernatant for the DNA damage assay. The number of passages of the cell line is 19. The cells cultured in T75 flasks were growing well and a little fast than before. Some people said that it is not good if cells grow too fast, is that true? Could this be the reason for the sudden death of my cells?
Thank you so much!!!!!!
For my other experiment, cells can live well in the medium without FBS. I just incubate for 24 hours.@Chunlong Zhao
I would suggest to repeat the experiment. And check the cells frequently. Maybe cells are infected
Hello Xintong Liu
One possibility I could think of was the medium without FBS as Chunlong Zhao pointed out. Sometimes cells cannot withstand serum starvation for long hours thereby triggering apoptosis and reducing cell survival. In such a case, serum starvation may be carried out in medium with low serum concentration (relative to normal growth medium), typically 0.1–0.5%, but sometimes as high as 1-2% or as low as 0.05%. Low serum concentration in the culture medium will help cells to remain healthy but not proliferate. But you have mentioned that cells can live well in the medium without FBS for 24 hours.
Then there remains another possibility of people who say that it is not good if cells grow too fast, to which I agree too, because excessive growth is likely to play a major role in the development of senescence, which occurs in many types of mammalian cells, and possibly it could show in porpoise too, which is a marine mammal. Unchecked cell growth leads to senescence, and this could be one of the possible explanations for what you have observed.
Are the fibroblast cells growing larger in size too? Have you noticed it? Increase in cell size starts diluting critical factors that are important for many cellular functions including cell division.
Best.
Hi Malcolm Nobre
Thank you so much for your useful answer!
Actually, I did observe that cell size increased with passage increase. I will check it again.
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