THP-1 cell culture problem: Cells die after thawing - (Jan/24/2008 )
I've been trying to grow THP-1 cells for a while but they die after a while. I'm using RPMI media with 10% FBS and 50uM 2-mercaptoethanol as suggested by ATCC. I'm keeping them below 1 million cells/ml. I tried several vials, and the cells are viable (Trypan blue) after thwaing, but they start dying after 2 days and eventually all die. We also received THP-1 cells from another lab from Europe, but they also died. I feel horrible since it's a pain to get cells form abroad. Can anybody give me some advice on how to grow them, or what might be going wrong. I know THP-1 must be easy to grow, but I'm not having any luck.
thanks
Hi,
i think you doing some mistake in reviving protocol can you tell me in detailes, because iam also using THP-1 cell line and many time i reviving from liquid N2 stock.
thanks
Thanks! I thaw one vial in a water bath at 37C and as soon as it thaws I transfer to a 15 ml tube and add 10 ml of RPMI-10% FBS media. I spin down at 1200 RPM for 5min, then I aspirate the supernatant and resuspend in media. I put in a T-25 flask standing.
First prepare the RPMI already in a tube.
Second don't thaw the cells completely in water bath. Take them out, when there is some ice left. They will thaw until you are at the bench.
Third add first some medium into the cryo tube before you transfer the cells to the prepared 15 ml tube.
When you thaw and resuspend your THP-1 cells, place them in a T25 flask which you then KEEP UPRIGHT for at least a week. Then lie the flask down as usual and don't attempt to pass for at least another week. They should recover fully with this treatment.
thanks