I am working with HepG2 cells for the first time. The culture is less dense in the middle of the flask and more dense at the periphery. Can some one please tell me if stage seen in the image is a good point for harvesting cells?
Hello Amrita Sharma
For HepG2 cells, 70-80% confluency is a good time to harvest these cells. As per the image attached, the cells are not yet ready for harvest. You need to wait till the culture is 80% confluent.
Best.
Can you also please tell me that 80% confluence should be in the middle or periphery, because the culture looks very different at different spot. Thank you Malcolm Nobre
Amrita Sharma
80% confluency should be all over the substratum. You need to re-seed the cells and ensure that they are distributed equally all over the substratum.
HI，maybe that because cells is aggregate when you pass them? To trypsinased cells into single cell. I normally wash cells twice with PBS, then add trypsin, keep monitor cells in microscope, flap the flask when they are ready. add cell culture medium, and give it a centrifuge. suspend cells with only 1ml medium, and transfer them to a cell culture dish. mix cells with a 1ml pipette, check cells in microscope each 20 times mixing, until more than 90% cells is single cell. if there still have some aggregate cells doesn‘t separate, then transfer cells into a 50ml tube, and add 30ml medium in it, let tube stand for 5-10min to let the cluster cells sink to the bottom, collect the upper cells and pass into a new flask. hope it help.
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