Adhesion of the cells when preparing for flow cytometry

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ChrSag
ChrSag's picture
Adhesion of the cells when preparing for flow cytometry

 Hello,
Lately I have experienced that cells have been adhering to each other when preparing for flow cytometry.
I am working with PBCMs and the first time it happended was after stimulation with PMA+Ionomycing. The adhering happened when I was adding Brefeldin A and monensin. It seemed to be the monensin making the cells/medium sticky.
 
Today I was going to label the cells with CellTrace, but after I had incubated the cells, centrifugated, discarded the supernatant and wanted to resuspend the cells in the residual volume; all the cells was gathered in a lump, making it impossible to resuspend. I had a concentrated suspension with cells and PBS for staining(2mL) and I added 4 uL Celltrace, which was dissolved in DMSO according to the manufactorer. It was incubated for 20 min at 37*C.
I tried centrifugating the cells again, with no luck. I also tried adding EDTA making a final concentration of 2mM with no luck.
Has this ever happened to anyone of you? Is there any way to prevent the cells from getting sticky when using celltrace?
 
Any advice is hugely appreciated!