I've been using a dextramer reagent to stain antigen-specific T cells in mouse lymph node cells. The stain works great, but it always clogs the FACSCalibur after about 10-20 tubes. I follow their procedures: the cells are strained in 40uM mesh, resuspended in PBS (not fixed) and read within two hours. I co-stain with CD8a and CD19. I know it has something to do with the dextramer because I divide all the samples in half, one gets stained with the dextramer protocol and the other half for a simple surface stain with other markers and it never clogs. But I can't figure out why this is happening. Can anyone suggest anything?
Have you tried adding in some protein (FBS or BSA) or a tiny bit of surfactant to your running buffer? Dextran polymers can aggregate and/or crosslink with each other, forming large complexes or potentially entrapping multiple cells. Those two phenomena might explain your clogging.
The only other suggestion would be to read sooner, use a plate sampler/HTS with rigorous mixing or strain immediately before putting tube on the FACS machine.
thanks for the tips, I'll try adding the protein to the final sample prep.
Try using 2 mM EDTA in your buffer that should keep cells from clumping and you should have a smooth flow in the column
Cloggin of the FACS after 10-20 tubes, I think could be due to dead and lysing cells. When cell release their DNA the DNA stick to the cells and make cell clumps. I just been to Cyto2013, and got the advice to try and add DNA'se to the resuspension buffer- I have not tested that, but is sound reasonable.
But filtering the cell suspension before FACS is always a good idea!
Dextramers does not stick cell together, not positive nor negative T cells, or other cells, that is easy to test. If Dextramer stick the cells together, the positive antigen specific T cells should give a higher degree of doublets, which is not the case, neither any other cell population seems to generate doublets in the presence of Dextramers, compared to in the absence of Dextramer reagents.
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