Hi, I am new to culturing 293T cells and was told that this cell line is particularly vulnerable.
I've received healthy stock from senior and have no problem growing them until when I keep stock and try to revive them, two attempts failed to revive cells (very little cells attached and looked very unhealthy).
My protocol for keeping cell stock:
1. Trypsinize 70% confluent 293T cells in T75 flask with 2ml trypsin, after all cells detached add 8ml fresh media and resuspend, count cell number and viability ( often >90%), spin down cells (1500 rpm, 5 min, room temp), discard media, add 700ul stock solution (10% DMSO in FCS) and resuspend with tips, transfer to cell vial, immediately stored in -80C freezer.
*One T75 flask cells for 1 vial of cell stock (about 3 - 5 millions cells)
My protocol for reviving cells:
1. After 2 days of storing in -80C, I try to revive to see if it works.
2. Melt one vial of 293T stock (about 3 - 5 millions cells) in 37C water bath, transfer cells to 10ml fresh media, spin down (1500 rpm, 5 min, room temp), discard media, add 12ml fresh media and resuspend, transfer to T-75 flask and incubate at 37C, 5%CO2.
These protocol are established one in my lab, I have also used the same protocol to keep stock and revive TZM-BL cells, it grows very well in contrast to 293T cells. So it makes me confused.
Please if anyone has the experience let me know which step possibly went wrong, or which step needs to be extra careful for a beginner like me.