Hi,
I'm trying to stain for actin in Jurkat cells the staining steps involve:
1. fixing cells in a 96 V bottom plate with 4% PFA
2. centrifuging at 1800 rpm for 3 mins
3. resuspending in 0.1% triton (I've also tried 0.05% triton) and leave for 5 mins
4. spinning cells at 1800 rpm for 3 mins.
5. resuspend in phalloidin and leave in the dark
When I check for pellets after step 4, they are either very small or there aren't any pellets. When I look at the cells on the flow cytometer, I have a huge amount of cell loss. Initially, I have a range of 500,000 - 150, 000 cells (depending on what I'm looking at) but after the triton step and spinning, I seem to lose the pellet and the flow cytometer only records under 200 events.
I've tried to reduce the centrifuge so its 1300rpm and 5 mins and I've also reduced the triton concentration to 0.05%. It hasn't made much of an impact. Any ideas?
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