I've been having trouble culturing LNCaP cells. We got the cells from ATCC and I've been culturing them in RPMI-1640 with 10% FBS and some antibiotics (1x Pen/strep). The first two passages went fine, but in this third passage, some cells have started to detach from the plate (100mm) two days after media change, leading to an accumulation of cell debris. The media also changes from orange to pink within a day of changing.There are still quite a lot of cells growing on the plate, the amount of cell debris is concerning me. For each passage, I trypsinize the cells for about 2 minutes and spin them down to remove the trypsin. What can I do to prevent this detachment and debris accumulation and what is the reason behind this? Thanks!
Most likely, cells are being "over-trypsinized" under the conditions used. The LNCaP cells attach to cell culture plate relatively loosely and do not require a 2 min trypsinization. Try reducing the time and use trypsin at room temperature or 4 degree C. Wash cells extensively to remove remaining trypsin.
...might the pH be the reason of the cell debris formation? I would check the pH of the media and also use flask/plate of a new stock. One time I got pink media because of a flask and the problem was solved just moving cells to a flask of a new stock. Good luck ;-)
LNcaP has poor attachment strength to substratum. Article The Blebbishield Emergency Program Overrides Chromosomal Ins...
Trypsinization should be super quick and wash with complete medium. The media becomes alkaline likely because cells die due to over trypsinization as suggested above by Dr. Divaker.
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