I've isolated human splenocytes. The tissue was dissociated, cells were spun down, red blood cells were lysed, and cells were washed a few times and filtered through 70um mesh. During processing, a large clump is usually produced and gets removed. Cells get frozen in a freezing medium composed of FBS and 10%DMSO.
When I thaw the cells, I notice a few clumps. I wash the DMSO off of them, and a few more clumps appear. I generally filter them once more through 70um mesh at this point.
The cells are still forming clumps that refuse to disaggregate even with pretty vigorous vortexing or pipetting. I've tried using trypsin digestion and collagenase type IV digestion (300U with CaCl2) to disaggregate, and the clumps seem to be pretty resistant to it.
Has this happened to anyone else? How did you solve the problem? I'm losing the vast majority of my cells to these clumps.
EDIT: problem was patient-specific. Resolved with high concentration collagenase digestion followed by serial trypsin digests (1000U/ml 30 mins, then 0.25% with 0.53mM ETA 3x5min).
Suspend the cells in SCSR transport mediium available from www.noninvasivetech.com. Pass the cell suspension after vortex through an 18 guage needle. Spin down the cells and resuspend them in SCSR transport
Can you expand on this a bit? I don't know that fecal sample preservation media is something I really want to use on precious donor-derived splenocytes. Is there a reason for using this - e.g., proteases in the media?
SCSR transport medium is a preservative for cells in general. Please refer to a paper on blood cell preservation in the October 2016 Plos One from investigators from PEI, Canada.
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