plating cells for transfection - (Feb/07/2006 )

I have been trying to perform the same set of transfections on 5 different cells lines. My problem is that one cell line, MCF-10A(Ras/erbB2), grows fine in 150cm dishes but when I plate them in 12-well dishes (at 4x10^5 cells per well) they do not look the same. The majority of the cells stay rounded up and those that attach barely open up. I figured that if I was the problem that I would be having the same issue with the other four cell lines, but I'm not. Also, after I count the cells and plate them in the 12-well dish I take some of the left over cells and plate them in a 150cm dish to keep growing. But these cells also grow awful. So it's like if the cells in the 12-well plate look bad then the cells in the 150cm plate will look bad too. Any ideas or am I just going crazy??

Have you had this problem over and over? And if so, if you just keep growing your cells in 150 cm (that's extremely large btw ;-)) they keep looking nice?

Have these cells been in culture for a very long time? Maybe they're too old. Try the procedure with a fresh batch of cells from cryo (have them in culture 2 weeks before trying).

thanks for the advice. my cells have only been in culture 4 weeks. i have this problem once in awhile, it's not consistent but it's enough to drive me bonkers. and yes, 150cm plates are large, but i need a massive amount of cells on a weekly basis you can only imagine the amount of media i go through...

i think the problem may stem from how many cells i'm plating per square cm since i can passage cells just fine into 150cm plates but not into 12-well plates. i did calculations last night and i'm plating 10-fold more cells per square cm. in the 12-well plate than the 150cm plate. so i think i will plate less cells and them sit on the plate for two days before i do my transfections.

thanks again!