I am doing cell culture of THP-1 cell line, at the first culturing and before incubation, 90% of the cells were dead because they were not stored in liquid nitrogen but in -80 C. How can I proliferate the 10% of live cells in the presence of 90% of dead cells in the same flask?
Hello Ahmed Al-Omar
First and foremost, you need to remove the dead cells from the flask because these dead cells may hinder the growth of the remaining 10% live cells. So, you need to centrifuge the cell suspension containing both the dead and live cells at low speed ( 100g for 10 mins) in order to separate the dead cells from the live cells. The live cells will settle at the bottom of the tube. You can discard the supernatant which will contain dead cells. Resuspend the live cell pellet in fresh growth medium and plate the cells in 24-well plate or a smaller Petri dish because the cell density is low and so the surface area has to be minimized to enable the cells to be in close proximity to each other. Just leave them in the CO2 incubator for a few days (2-3 days). Keep observing the THP-1 cells everyday for signs of cell growth.
I hope this helps.
Good Luck.
All I would add to Malcolm Nobre's answer is that I underlay the cell suspension with 1 ml FBS to act as a density cushion. The live cells sediment through the FBS layer and the dead cells/debris does not. I actually do this routinely whenever I originally thaw cells that have been frozen, be it from liquid nitrogen or -80oC.
Gary Lee Gilmore Malcolm Nobre Thank you very much for your answers. It is really helpful :)
I would remove the dead cells first. Low-speed centrifugation (800 rpm, 15 min) works really well. Live cells will be at the bottom and dead cells in the suspension. Discard the supernatant and then count the live cells. Resuspend the cells in fresh media at a density of at least 300,000 per ml. Hope it works.
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