I have my protocol for fixing cells, however it tells me to fix at 2 million cells per ml with 2% PFA. I am wondering if i can use a higher concentration of cells to fix with as i normally have ~100 million cells per ml to work with. All the diluting and splitting of my cells ends up losing quite a bit and i need all the cells i can get. So far i have had no luck with finding an answer to my question on the net. I need to fix my cells then permeablise them for a phospho-flow analysis.
Thanks in advance!
Hi! probably try to fix at 5 million / ml. i'm not sure that it has to do with your protocol 100% cause in all cases you'll loose cells.
I routinely fix cells at a final concentration of 30-50 million cells/ml (using CytoFix/Perm from BD), sometimes more. To minimize cell lose (due to clumping) at such a concentration I would recommend to resuspend the cells in a small volume of PBS first, before adding the PFA. When adding PFA directly to the pellet you increase the change of cross-linking cells to each other causing clumps. Good luck, G.
- Badges
- Science method