Cloudy/Turbid media after subculture - (Apr/11/2011 )

Hi,

I have been having a problem every time I subculture SH-SY5Y cells, the media turns turbid overnight, and the cells are not attached, I have tried a couple of times over the past 6 months, made fresh media and PBS, when I change the media, the cells are fine, however after subculturing them, they do not attach and the media is cloudy on the next day, is it a contamination, or could it be due to overtrypsinization ( I add 2 mL Trypsin to the 75 cm flask for 2 minutes, and then add 2 mL of 10% FBS DMEMf12 media, 1% Penicillin/Streptomycin)? this has been going on for months now,,,,,,, and altough a few times I did not have this problem, and I can't figure out what is going on

First of all - have you checked if it is bacterial or yeast contamination?

There are two populations of cells in SH-SY5Y cultures - some of which are attached and others of which are suspension cells. For proper maintenance of the line of you should be subculturing both when you split the cells. Perhaps this is what you are seeing.

Thank you Bob,

It was contamination after all, one of the flasks was fine, but then got turbid again, I got rid of all the cells and got new cells, my labmate advised me not to use the Trypsin we have for subculture, since contamination only appeared after the subculture, but not when I changed the media for the cells.

I would get rid of your medium and PBS too. Cleaning down your pipettors and anything else you use routinely in cell culture is also a good idea.