Hi everyone!
I am currently trying to differentiate and culture murine eosinophils from bone marrow cells (with FLT3-L, SCF, and IL-5) according to Dyer et al., 2008.
After 18 days in culture I wanted to check eosinophil marker expression with flow cytometry, but I kept loosing cells, most likely during centrifugation.
Based on my work with T cells, I usually centrifuge at 500xg and 8°C for 2 (plate) or 5 (tube) minutes. My cell input was enough for a visible pellet. After transferring cells onto a plate, 2 min at 500xg gave me a nice pellet. After staining, I used the same setting and did not get any pellet. I centrifuged again for 2 min, but at 650xg. This gave me a pellet. After washing, I centrifuged again at 650xg, but did not get a pellet, so I increased to 850xg.
When I acquired my samples, I ended up having a very low event rate, so I assume that I lost a lot of cells in the staining process. I know that it is normal to loose cells during wash steps, but this seemed different.
I am very new to working with myeloid cells and especially eosinophils. Can anybody help?
If you do have functional granulocytes in your differentiation mix, it's likely that you're losing them to activation. They de-granulate, attacking the next best thing, like the tube wall. Then you have cell death, genomic DNA release, which makes the solution viscous and stringy, impossible to centrifuge. (watch for that!).
It's not so much a matter of "settings" than about avoiding anything that could startle the cells. Impurities in reagents, less than perfectly clean plastics, mechanical agitation, any delays in processing. Look at your process, and try to identify anything that could cause your heavily armed and extremely jittery cells to go haywire. Your staining protocol is probably way longer than it needs to be. Determine experimentally which are the shortest incubation times you can get away with.
Finally, consider Giemsa staining on slides instead of flow. It will have less of these problems.
Thank you, John! These tips are very helpful, because you're right. My handling has been anything but gentle and I indeed observed that the cells/medium was already very "fuzzy"/grainy before I even took a sample for staining. So I guess my cells are already dying in culture?
I will check my differentiation protocol and reagents and my technique again.
Hi Michelle,
Many of the protocols I have seen almost all stay at 300 to 400 xg (RCF).
I agree with other answers here. You should always consider what is happening upstream that may be causing you issues. Granulocytes are particularly difficult because they degranulate. Interaction with certain receptors (perhaps from your experimental treatments) can also be part of the problem.
Here is an example protocol:
Article Bone Marrow Derived Eosinophil Cultures
I would stick to lower RCF speeds and use longer times whenever you see viability issues in these types of cultures. Another thing you can try is live/dead staining with something as simple as Trypan Blue at any step you think may be causing the problem to identify which step is the problem.
Best of luck!
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