I have counted different cell lines like HeLa, LNCap.FGC, PC-3, CHO-b using a haemocytometer on T-25 and T-75 flasks. But even when the flasks are fully confluent, or 90% confluent, I get a very low cell count form flask. I usually trypsinize the flasks, pellet down the cells and add fresh 1-3ml of medium depending on cell density before counting. I make sure the flasks are always completely trypsinized with hardly any cells left behind after trypsinization. I also pipette and mix the cell pellet in the 1-3ml of medium thoroughly, so that there are no clumps. But I have got very low cell counts each and every time. Why is this so?
Before 'pelleting down the cells' (by centrifugation, what speed?), you have to stop the trypsine action by adding full medium (10% serum). Is it the case?
Yes, I do add medium with 10% FBS and I centrifuge at 800 rpm for 5 minutes at 22 deg C
check if you have cells in the supernatant after centrifugation if you have a microscope near by?
There has to be some kind of mixing problem of the cell pellet. We need to dilute one step more (from initial 2-3 ml suspension) to avoid crowding of cells in the haemocytometer. Pl also check that cells are not broken as suggested by Anne.
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