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Losing cells during intracellular staining method! - (Jul/16/2013 )

Hello! I hope you can help me out with this one:

I am trying to detect a nuclear protein in my mammalian cell samples using flow and an intracellular staining method, which involves fixing and permeabilzing the cells before staining them. However, at the end of the procedure I lose all of my cells, probably because of the many washing steps involved. Can anyone give me useful tips to prevent this from happening? Many thanks!!

This is the protocol I use:

1. Harvest cells, count cells and spin them down (2000 rpm, 5 min)
2. Resuspend cells in PBS to get 1x10e6 cells/ml/sample
3. Fix cells by adding 40 µl of formaldehyde (final concentration 4%)
4. Incubate for 20 min at RT
5. Spin cells down (1000 rpm, 5 min) and wash 3x with 1ml PBS (keep on ice)
6. In last washing step leave 100µl of PBS (on ice)
7. Permeabilize the cells by slowly adding 900 µl of 100% cold methanol to pre-chilled cells (final concentration 90%)
8. Incubate for 30 min on ice
9. Proceed with immunostaining or cells can be stored at -20°C in 90% methanol
10. Spin cells down (1000 rpm, 5 min) and wash cells 3x with 1ml PBS
11. Block cells with 500µl of 0.5% BSA in PBS for 1 hr at RT
12. Add primary antibody to each sample
13. Incubate for 1 hr at RT
14. Spin cells down (1000 rpm, 5 min) and wash cells 3x with 1ml PBS
15. Add FITC conjugated secondary antibody in 500 µl of 0.5% BSA in PBS to the cells
16. Incubate for 1 hr at RT in the dark
17. Spin cells down (1000 rpm, 5 min) and wash cells 3x with 1ml PBS
18. Resuspend cells in 0.5 ml PBS buffer and analyse on flow cytometer


Looks like it really is due to the many washing steps. I also often perform intranuclear FACS stainings (although I wash a little less than you do and follow a different fixation protocol) and have exactly the same problem. I used to think that maybe some cells get stuck at the walls of the tube during centrifuging. I tried to play around with centrifugation speed (see this thread ) but that did not help.
Other than being r e a l l y careful when removing the supernatant (rather leave too much than take cells away accidentally) I never found a real solution to the problem. By being extremely careful, I managed to get slightly better over time but I still lose a lot.