I am growing Sf9 cells in suspension culture and while they are growing ok, their viability has dropped considerably and so there is an accumulation of dead cell debris. Do I need to remove this? And if so any advice for doing so? Will the cells still grow ok with the debris and then I can dilute it out with subsequent passages?
Hi Rachael,
I agree with Fernando Arosa. If viability drops below 80%, spin the cells at 300 RPM, discard all the sup and suspend the cells in SFM gently. Check the viability before diluting further for growth.
Good luck
I agree with Fernando but you have another option is to dilute the cells to a very low number and reculture it in new plate for example one cell per well in this way you will get a purify and active cells without dead cells
It is better to remove the dead cells because it will affect the viablity of your cells may be drive your cells to apoptosis and debris will increase the viscusity of the medium in same time so it is better to remove it
Good Luck
Farooq, UCL
I agree with Fernando, centrifugation should be done very gentle and only in the bucket rotors, with low speed, not more that 100 RPM. Bucket rotor (no fix angle rotor) is very essential! good luck!
Hello Rachael
Follow either one of the two:
1. Centrifuge the cells to 300g for removal of debris
2. Just remove the supernatant, wash with PBS and add fresh medium
Best Wishes
Hi Rachel, I think you got your answer already, from the previous posts. I would just like to add that also, for your experiments, since cell debris contain biomolecules, such as proteins, you might get a wrong estimation of total protein levels in your "living" preparation. Many researchers normalize specific protein levels to so-called "housekeeping" proteins, such as tubulin or actin, which serve as an estimation of the total amount of cells that were originally in the lysates. If you keep cells debris, you'll increase the error in this estimation by including protein from dead cells, underestimating some experimental effect you might be looking after.
Good luck!
You do not want to have a lot of dead cells (normal healthy shaker cultures should be >95% viable cells, and preferably 99%), and their presence means something is wrong (infection with a virus/bacteria/fungus, old media, improper storage of media, incubator temp. too high, shaker speed too high). The cells require L-glutamine and it is temp. and light sensitive, if you repeatedly warm the same bottle of media the L-glutamine will break down to ammonia and that is toxic to the cells (but media made with glutamax is fine). And media with L-glutamine has a limited shelf life at 5oC for that reason (1 month after the L-glutamine is added for Express Five media). The cell viability can also drop due to the cells being shaken too hard for the size of the flask. The thing to remember about shaker flasks is that the larger the flask is in diameter the higher the shear force the cells will see at the outer edge of the flask "at the same rpm", and too high of a shear force will kill the cells (this is even more of a problem with baffled flasks and serum free media). So cells in a 250 mL flask at 100 rpm will see less shear force than cells in a 1000 mL flask at 100 rpm. Try going back to T-flasks to get viability >95% (after the cells attach, just remove the media with the dead cells). Then after expanding the cells in T-flasks, transfer the healthy cells back to shaker flasks starting at around 100 rpm for a 250 mL flask at >0.5 x 10e6 viable cells/mL.
i agree with Fernando Arosa. Try to remove debris by doing centrifuge with low speed (300rpm) or wash with PBS is the best option.....
May consider Celltrics in appropriate pore size which fits your cell to filter out debris.
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