Dead cells or cell debris are supposedly to be lighter than live cells, and therefore the most recommended procedure is to centrifuge and remove supernatants.
Recently I used centrifugation at 200g for 3min to pellet live cells, but found that 90% of the cells showed dead by trypan blue staining.
I also read other's recommendation on internet and collected medium without disturbing dead cells/cell debris on the bottom of culture dish, but still got the same result.
This is most frustrating....
Somehow I felt confused:
if the dead cells actually sink to the bottom while live cells remain in suspension, how could centrifugation separate them by pelleting live ones?
Or maybe I could stall the cells in falcon tube and wait for debris to fall down with gravity?
Try using Histopaque or ficoll for density gradient centrifugal separation of lymphoblastoid cells. Dead cells will have different density than live ones.
If your cell viability had been above 50-60% they could still be savaged. In my experience, dead cells normally form a large cellular aggregates, or clumps, that appear 'sticky' after pipetting. Meanwhile, live cells will remain as single cells in the suspension. You can savage the healthy live cells by simply avoiding the large clump of dead cells during pipetting.
In your case, 90% dead cells means that you will have a large 'dead cell clump' with virtually next to 0 live cells in your suspension. It's easier to thaw back-up cells from -80 or liquid nitrogen should you have any.
Besides, the release of apoptotic body from the dead cells (90%) were likely to induce apoptosis in the remaining live cells. Although they may appear trypan blue negative under the microscope, your live cell population could be pre-apoptotic. In this case, it is not advised to savage the 'live cells' that is likely to ruin your experiment/time/effort.
If you still wish to proceed, savage your live cell and try growing them in a smaller tissue culture format (smaller plate/flask) and leave them at higher cell density. Hopefully the cell-cell contact can put them back to track. And change your media frequently.
Hope this helps, good luck.
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