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293T cell line, how to prevent cell clusters? - (Mar/23/2006 )

Hi, anyone here has the experience of 293T cell line? I now have problem to seperate them into sigle cell when i splited them. They are easily to make islands with two or three or even more cells. I tried to leave them in trypsin for a longer time, from 1min to 5min, but it did not help. it seemed to be even harder to seperate them when i left 5min in trypsin. I also tried to separate them harshly with pipet, but i was told it's not good for the cells ( and also can not solve the problem perfectly). Now i really have no idea how to do with them. Anyone can help me? Thanks very much.



I would leave them in the trypsin until the cells just detach with a tap on the side of the flask, and then add medium and pipette up and down about 5-10 times, You can be reasonably vigorous for this, 293s are pretty tough.