Hello,
Today, as described above, I want to ask whether dead cells(Cells dead because of siRNA and DNA cytotoxicity)
affect RNA and Protein concentration. I always make sure all samples
have same amounts of RNA and Proteins for reliablility of research.
But i realized that I do not shake gently cell culture tools in washing step.
In washing step, I pour serum-free media or PBS to cells and then
just lean culture tools to remove such solutions.
So I am worried that dead cells that should be removed in washing step
can still be remained. In this case, Does RNA and Proteins from dead cells
are involved in measurements of RNA and Protein concentration?
I think that whether shaking cell culture tools or not is not critical for removing dead cells
described above (But good if conducted) in washing step because cells dead from siRNA and DNA cytotoxicity will be removed by suction and leaning described above
is also action of shaking culture tools.
I want to know opinion from others.
Thank you!
Cell attachment is different for different cell types. What cells do you culture? If you want to remove dead cells - look at the microscope first and check whether some cells are rounded and detached. You might need to shake them a bit to help detachment. You also need to use other methods to confirm apoptosis if you want to estimate the amount of dead cells in your culture - use fluorescent staining. If you have too many dead cells it will affect the level of your RNA and protein as it might degrade due to RNAase/protease activity etc.
Thank you Olga!
My question above is about HUVECs.
How do you think about my method of removing dead cells?
I appreciate again
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