Every time I wash cells, they began to detaching and get circular for some minutes and after replacing the medium they reattach again, does anyone please have idea to stop it?
I have tried to keep PBS in 37.5 degrees of Celsius and also I have added 1mM of Mg and Ca. a friend of mine said maybe it's due to the passage number of the cells.
Hi Haref, I do see the same phenomenon in case of 293T, I am thinking this is because of sudden change of pH from lower (media colour yellow,over grown, pH 6) to normal pH 7, PBS and/or fresh media.
dear Uchurappa Mala
my supervisor said it can happen due to low temperature as I kept PBS in 4 degrees of Celsius. and then I tried to warm them up before washing the cells, but it did not work. so did you tried to use it when they are in room temperature yet?
best regards
Aref, generally we keep everything at 37 degree celsius then only use for cells. Then take fresh vial of MCF7 and thaw. In general after so many passages there are chances that cells can change their morphology and other physiological properties.
For sensitive cells, the brief time without bicarbonate buffering is enough to change their morphology. Though more expensive, try using CO2 equilibrated serum free media.
Try different Cell culture flask? Is your media HEPES buffered? Some labs grow breast cancer cell lines in HEPES buffered media, to protect cells, but they inadvertently select for pH sensitive cell lines. Buy new cell line from atcc. Your time/consumables/money wasted is not worth a bad cell line. I know the cost has double, use to be around $200 now $400, but it still is critical that you have a good cell line. Be sure, buy your own cell line and keep track of passage number and freeze a bunch of the lower passage number for future work. MC7's stay on flask and don't come off like you describe, unless they are overgrown and gone basic, in which most cells will.
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