I seeded MEFs (passage 5), mitomycin C, or UV treated MEFs ( passage 6) in a 96-well plate at a density of 40, 000 cells/well, since I wanted the wells to be very confluent. I always see a lot of debris/junk on my plate, and the cells are never confluent, even when I seed a lot of cells. These MEFs will be used as feeder cells for co-culture. The debris/junk is really annoying and I cannot perform my experiment until this is solved. Any suggestions would be great. Thanks.
PS: I coated one time with Gelatin or Poly-lysine, and it seems to not be helping at all.
Looks like dead cells to me. Were those prepared fresh and then passaged 5x or have they been frozen in between? Have you tried to wash them a couple of times and noticed whether the debris returned? 40,000 seems indeed pretty damm confluent to me, maybe thats whats stressing them and therefore they die? I would wash the original falsk extensively before trypsinising and plating them. Have you tried different densities and checked whether there is less debris at lower cell densities? Also, check your medium. Good luck.
Hi,
1) This can be due to the matrix, mit C treated MEFs are to be seeded on gelatin 0.1%
2) can be due to high density seeding which results in peeling of the attached cells hence forming debris .
3) If the Mitc or irradiation is not done properly cells do not adhere and go through senescence.
Yes, compare different densities because a lower number may give you an optimal spread. Always pre-treat with 0.1% gelatin and don't hesitate to leave it on there a long time and put at 37 degrees if you have the time. When I made primary MEFs and expanded them, I noticed they just don't do well after about P4 stage, so P5 wouldn't have worked well for me either.
Also, make sure to use flat-bottom 96-well plates, a round-bottom doesn't allow as even of a spread.
Thanks a lot for all your answers. I will wash my plates with PBS twice before trypsinizing them and also try different densities of cells. However, I still want the cells to be as confluent as possible.
I will use early passage of MEFs, at passage 4.
I am not sure that, must we coat the plates with Gelatin to culture MEFs? When I expanded the primary MEFs, they adhered and behaved very well without any coating. Thanks.
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