Dear researches
I see white spots with the naked eye in the culture flask of CHO cells. in the microscope, the white spots are large aggregates of adherent rounded cells. i also notice that the background of my cell-based assay is increasing and my controls decreasing in value.
have anyone experienced the same? and what can be the cause of it?
Hi May,
It happened also to me. My hypothesis is that during the trypsinization you do not separate all the cells and so when you plate them in a new flask these cellular aggregates will form these spots. Did you select them using any antibiotic?
While trypsinizing your cells try to use Tripsin+EDTA that brakes cell clumps and resuspend the cells well before planting them in the new flask/plate.
Hi,
As the fellows said the cells should be separated and detached from the surface of the flask. In order to do that, you need to dispense the suspension cells in the flask by repeated pipetting onto flask surface and tapping gently. Use Trypsin already has EDTA and incubate in appropriate time.
Best of Luck
Hi Mai
Look carefully under the microscope, do they look as cellular clumps or do you see something else also, like filamentous outgrowths or something like that. If so then, mate, your cells might be contaminated with Algae/Fungi, in that case, dump the culture and start afresh. If you are sure it is not a contamination try triturating them more when you trypsinize them next time on the dish itself. Also, you can try and avoid taking the clumps forward by light trypsinization, that way the clumps will stay behind and you can passage the single cell suspension.
Cheers
Abhishek
Dear all
thank you for taking your time to solve this
when i split the cells, i wash 3x with PBS and add 3 ml trypsin to the 75 cm2 culture flask and incubate for 5 min, tap the flask, add medium and spin the cells down to resuspend them in new media. They look separated under the micrscope. I didnt find any filaments in the white spots and the media contains pen/strep and zeocin
my problem is also the increasing background of my cell assay with increasing passaging which is not a problem for our EBNA293 cell line
Hi,
I think these spots and accumulated cells due to the confluence of your originated flask before splitting. the confluence has to be between 70-80%.
when you be in the process of trypsinisation after tapping and adding 10ml the media (serum media to inhibit the trypsin) in order to pipetting down the cells in the flask, try to not transfer it into tube for centrifugation but instead transfer only 1-0.5ml of the cell suspension into new 75cm2 flask media and add serum media up to 20 ml.
This process is a appropriate for only a confluent cells about 75% and above unless counted cells is required.
Best of Luck
Ameen
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