Hi,
We're having a bit of a problem while trying to find progenitor populations in bone marrow.
The protocol for CyTOF includes staining the cells, fixation, methanol perm for 15min, intracellular staining, fix and washes in 800g and Ir staining.
After washing the methanol, we had 5 million cells. However after Ir staining we were left with 3 million. Meaning we lost 2 million cells in the process. In addition, our CyTOF keeps clogging because of strange aggregates.
This feels a bit excessive and I was wondering where in the process we had it wrong.
Methanol is ice cold (-20C), added drop-wise, centrifuges were done in 800g.
Is it possible the cells were more vulnerable after the methanol and exploded in such speed?
Thanks
Mayan
We also had considerable cell loss during the staining process for CyTOF, and after testing several parameters including buffers, fixation method etc. the only things that reduced cell loss in our hands was :
- to work in V-bottom plates rather than with tubes
- increase centrifugation force to 1000 x g right after methanol treatment and for all centrifugations until the end.
We still had cell loss afterwards but this was really a lot less pronounced.
Thank you Julien, that's very interesting.
We thought we needed to reduce it. Did you also have aggregates before performing these steps ?
In my hands cell loss was largely due to the fact that there are many centrifugation steps (more than in FACS for instance), and I really compared lower vs. higher centrifugation steps and this appeared to be one major parameter that could reduce cell loss. I also thought 1000 x g was too high and would harm cells, but it worked like a charm and it might be even possible to go higher (I also did not mention that I began working at 800 x g, just like you, and I spun at 800 x g for all centrifugations until the methanol step).
I don't remember having big issues with aggregates, but I was working with human PBMCs and not bone-marrow cells so this was a bit different from you.
However, before I figured out that increasing centrifugation speed and working in V-bottom plates reduced cell loss, I tested if treating cells with DNase could help. The reason why I tested this is because I was suspecting dead cells (following the cisplatin treatment, which stains dead cells but which also induce cell death) to activate cells and/or to create some smears that would impair the process (trapping cells from the pellet, that would then move to the supernatant, for instance). Maybe that could be something you could test in your case ? Another thing we tested, but this was a pain in the neck, was to check the presence of cells in the supernatant after each centrifugation step, but I don't remember something spectacular from this and if I remember correctly number of cells found in the supernatant just began to increase after the methanol permeabilization.
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