For TUNEL assay and cell cycle profile analysis, more than half of my pellet gets discarded after washing and removing supernatent after 70% ethanol addition. The cell lines are MCF7 and MDAMB231. How i can avoid this critical failure ?
Most cells in the cell pellet are lost during "washing" step (suspension of cells, centrifugation, and removal of supernatant). If one is able to remove supernatant by sucking it (through a vacuum), the cell loss can be minimized.
Highest speed should be used at washing step and supernatant should be removed with the pippette of 10-20ul.
Beast of luck
I think that sometimes the problem is rather that the cells stick to the tube. Resuspending the cell pellet in ±100 uL full medium (with FBS in there to help block aspecific binding) before adding the ethanol resolved this problem for me.
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