I am culturing suspension cells (Jurkats) and they are in 96 well plate ('v' bottom). I have been noticing a lot of cells (>50%) being lost despite centrifuging them and aspirating the media from the side.
Centrifuge speed : 2000 RPM / 800g (tested a few this seemed to give the tightest pellet) for 5 minutes
Seeding amount : 100000 cells
Harvesting : Done at the end of the day
Amount Expected after washing : Close to 100000 cells
Empty the nutrient medium into tubes and do not throw it away. Also, the amount of PBS that you use to wash the cells, empty it after washing into the same tubes
After that, make a centrifuge and then return the precipitated cells to the plate containing the cells again after adding the new medium.
Thank you Ali for the reply, but what if I have 96 samples. It will take me a very long time to complete the entire process.
This is true, but at least you can collect the supernatant from repeated sample wells (same treatment and concentration) in one tube, and after centrifugation redistribute them evenly.
Try also to increase the centrifugation period and when removing the medium, withdraw it quietly and in stages with a multi-pipet and do not withdraw it all (if the medium is 100 microliters, for example, leave 25 microliters in the well and add the new medium or treatment substance to it with the calculation of the concentration difference.
Also, if the cells do not leave residues and the environment is clean in the wells, there is no need to wash them with PBS.
Try 2-3% FBS in PBS to resuspend and wash cells (if FBS can be ignored in your following experiments ) or at least use 2-3% FBS in PBS to pre-treat your 1.5 ml microtubes or 15ml tubes to make them "wet" or "slippery".
Hi P Lokamitra P Lokamitra
The solution might be a contradiction. I recommend to reduce the centrifugation-force to 200 or 500xg max. Jurkat are quite sensitive to sheer stress. If you have a solid pellet you have to pipet quite harsh to resuspend the pellet. By this you will easily loose 50 % of the cells having only cell debris left.
If you are working with cells a lot you should check-out Cellseeker (https://www.cellseeker.org/). This is an easy and free to use app to organize and inventory your cell collection online.
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