I found my U2OS cells were able to adhere to the dish after sorting by flow cytometry, but almost all died after two or three days.
Did I use the wrong culture conditions?
Complete medium: DMEM （gibco, C11965500）+ 10% FBS + 1% Penicillin-Streptomycin Solution
37℃, 5% CO2
Hello Hanfang Xu
If cells are exposed to stress, the consequence is cell death. During cell sorting, you could make some changes in order to minimize the impact on the cells. You need to keep your cells happy while isolating pure population of cells.
You should make sure that you sort the cells in a medium in which the cells remain happy. Usually a medium containing high serum concentration would be appropriate (around 30-40% serum). Cell culture medium buffered with a CO2-carbonate system would not be a good choice for pH-related reasons.
Another factor to keep in mind is the collection tube. Cells may be damaged if they hit the side of a collection tube. So ensure that the collection tube contains the appropriate medium and at the appropriate level so that the cells do not stick to the walls of the tube.
You should run cells at lower pressure as this will decrease the shear forces that cells experience thereby preventing the detrimental effects on the cells’ biology. Also, keep the sample pressure low to ensure that the core stream, through which the cells flow, is narrow and that its velocity is consistent.
Always keep your cells on ice in order to slow down the metabolic processes. But you should also know that mammalian cells become stressed and die quickly at 4°C as cold temperatures prevent cells from repairing any damage that may be caused during the sorting process. So, see to it that you choose the best temperature for your cells during sorting.
I feel that if you optimize the pressure, temperature, and components of your buffer solution, the cells may be less stressed and you may succeed in culturing the cells after sorting.
Best.
The only point I would add to the excellent explanation from Malcolm Nobre is that it is important to be aware of the size of your cells relative to the sorter nozzle aperture. It's been years since I have sorted cells, but I know that most sorters are set up for sorting lymphocyte sized cells, and if your cells are larger and you are using the standard nozzle, you can be shearing your cell outer membranes, causing cell death. I suggest you check the manual for your sorter, or talk with the sorting technician about nozzle size.
Recently, I used DMEM with 20% serum after sorting to solve the problem of cell death.
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