It is my first attempt with Flow cytometry (acquisition) and I am having a problem during the thawing procedure. I work with PBMCs and I have to manage with a pellet of cells which I cannot resuspend during the whole procedure.
At first I add pre-warmed medium FBS-RPMI into the cryovial, dropwise. After the first centrifugation, I add DNase, PBS, and FBS-RPMI. At this step, I cannot resuspend my pellet. It is a sticky pellet, probably of dead cells, which It cant be resuspended
at any step. So, I cannot proceed with the staining procedure.
Does anyone have any idea on what to do to overcome this?
Thank you in advance,
Elina
You are definitely lysing your cells and the released DNA is why the pellet is sticky and will not resuspend. The method you describe is unlike any thawing procedure I am aware of. The problem is that 10% DMSO is hypertonic, and the unfrozen cells will lyse under those conditions. You are slowly diluting your thawed cells with warm medium which will worsen the problem.
The method I use is to prepare a tube with cold medium, thaw the cryovial, immediately transfer the contents of the vial into the tube with medium and centrifuge 200 x g for 10 minutes. I typically underlay 1 ml FBS under 9 ml cold medium to cushion the cells as they are being centrifuged.
Miltenyi makes a product called MACSQuant Tyto Running buffer that contains a proprietary DNAse enzyme. It runs ~ $100 for $100 ml. I have found that if I resuspend the cells with this buffer and wash the cells by spinning them down again and resuspending them in Tyto Running Buffer, I have no issue with cells clumping - even patient PBMC samples that have been stored for over a decade at - 80oC.
Gary Lee Gilmore thank you very much for your immediate and detailed answer. Regarding your comment on the slow dilution, the truth is that the thawing protocol remarks the dropwise handling.
I will follow your Recommendation on the Tyto buffer
I would recommend not to freeze. Use fresh cells for FACS and use calcium and magnesium free PBS to wash. Avoid vibrations or vortexing.
Goodwin G. Jinesh unfortunately that is not possible. Samples are freezed in -80oC since 2020. Thank you very much for your kind reply.
- Badges
- Science topic