In our lab, some cell lines are dying and some cells were stay alive after trypsinazation. For example, A549 cells are normal after thaw them from -80. Subculture them and then we found them dead. But this situation is not depends on cell line because some A549 cells that are thaw from another stock are stay alive even after subculture them. We thaw PC-3 cells and after subculture we stock half of it and seed half of it. Seeded half was dead next day. So we we thaw other half from stock and at first they were alive. Subcultured them and they were dead. I will leave photos from DU-145 cell line that are taken from flask full of dead cells. Why is this happenning? Also we are currently growing some cell lines without any issue (PC-3, MDA-MB-231 etc.). But we saw this issue from some stocks even from this good growing cell lines.
Dear Okan,
You might wanna check whether they are all mycoplasma free. Cause suddent deaths as like your case after first passage always remind me this particular kind of contamination or else the quality of FBS but you had no problem with other cells.
You can find a PCR or ELISA based kits to check the contamination
Best
Yunus
Hey Okan,
Can I ask, whether you use warmed trypsin or cold one? You can also seed them after washing your cells thrice in PBS to get rid of trypsin in them.
Washing of cells:
Add Warmed PBS in the cells in a falcon tube (15 or 50 ml) and centrifuge them, Discard supernatant and add new PBS and centrifuge.
Regards,
Zubair
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