Hello
I am isolating bovine PBMCs and staining them with the cell violet proliferation dye for a proliferation assay. I also include an unstained sample. My unstained are alive even after 8 days of culture. My violet stained cells are dying even after 2 hours. I am using flow cytometry Canto and I can see totally no population ( only trash) in comparison to my unstained samples. Can the cell be killed if my dye is one month expired even after 2 hours or the DMSO added to the dye is killing my cells? Any clues ?
Thanks
Staining cells for proliferation with DMSO resuspended dyes can lead to cell death.
So try the following:
- use 3-10 times less dye than the maximum recommended dose
- do not incubate the cells with the dye for more than 10 min at 37C
- wash the cells twice with media after the staining
- recount cells and check viability
- plate the cells for culture
Good luck
What DMSO concentration are you using?
You have to bear in mind that, besides many other properties, DMSO acts as a carrier for almost any substance, so the dye will penetrate much more to the cell. In that sense I agree with Eric B. Brandt about using a lower amount of dye and extra washing.
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