My workflow involves creating fluorescently barcoded regions on a spleen sample. I then dissociate the sample in saline solution and take it to the flow core for sorting of the positive vs negative populations. We use a Moflo Astrios, and it also sorts into saline solution. I haven't been able to pellet down my cells for subsequent processing with trizol. Has anyone ever done anything similar? Is there a way to sort directly into trizol with this instrument? I think the volume post-sorting is too high which is why I can't see a pellet. Is there a way to reduce the final volume so it's more concentrated? Perhaps I should use another instrument? Honestly, any information for what I can do to isolate RNA post sorting would be very appreciated.
How many cells are you typically sorting and what's the volume your cells are suspended in post-sorting? These details will make it easier to solve your problem.
Sabine Strehl Thanks for responding! so when I did it last time, my population of interest was around 500k cells in 7.5ml. It was pretty dilute, and I wasn't really able to collect the pellet. The sorter can tolerate concentrations of 5x10^6cells/ml (which I've tried) but even that results in my final sorted sample being in a huge volume. Do you have any suggestions?
This is really a large volume for 500K cells. How long are you spinning your cells and at which rpm or g? May be a higher speed and/or centrifugation time up to 20 minutes might help. As you are isolating RNA right away cell viability should not be that much of an issue.
Another option might be to aliquot your cell suspension into to 1.5 ml Eppendorf tubes and spin them for 5-10 mins at 800 × g (or at maximum speed). Remember how you placed the tubes in the centrifuge and carefully look whether you see a tiny cell pellet on the outward pointing bottom (wall) of the tube, carefully aspirate the supernatant (even if you do not see a pellet) with a 200 µl pipette tip or carefully poor off the supernatant. Then resuspend the cells in each tube in an appropriate amount of TRIZol so that you can again merge the lysates in one single tube and conduct the RNA isolation. Instead of TRIZol, an RNA isolation micro kit available from various vendors might be another option.
Please remember, a that a typical mammalian cell contains 10-30pg of RNA, thus, depending on the RNA isolation protocol and the cell type, the maximum total RNA yield from 500K cells will be 5µg.
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