I hope you'd be able to share a few insights. I am attempting to perform a ChIP experiment using FACS sorted cell populations. After sorting cells were pelleted and snap frozen. And that's what I get as samples. The cell number in samples is quite limited. After pooling a few samples together I can expect between 300 000 and 500 000 cells. I then follow this protocol:
- Resuspend cells in freshly prepared 1% FA in PBS, fix them for 10 min at RT;
- Add Glycine, incubate for further 10 min at RT;
- Spin the cells
- Resuspend the pellet in ice cold PBS
I count the cells before and after spinning and somehow I manage to lose 90% of them during the spin. First I thought I didn't spin them fast enough, so I tried spinning them at 1000, 2000 and 5000 rcf. Then I thought I'm spinning them too harshly and tried spinning them at 100, 300, and 500 rcf. All to no avail. I have also tried resuspending cells in PBS with protein inhibitor cocktail instead of fixative. But am getting the same result. So frankly I'm running out of ideas and any thoughts/ suggestions would be much appreciated. In ideal world I would probably fix cells straight after sorting, but since all the samples are already collected that would mean a big waste of time and samples.