I have been trying to remove cell debris from my cell suspension after tissue dissociation, since I have to perform a single-cell.
I work with skeletal muscle and in the mice that I injure with CTX and deplete the infiltrating macrophages, my cell suspension has a lot of debris (specially tiny ones), even though it has a high cell viability (~90%). This doesn't happen in normal CTX injured muscle, where I have almost no debris and easy to be eliminated.
I have tried different dissociation protocols, use of magnetic beads, sorting of live cells and the MACS debris removal solution (that helps but still I don't have a good sample for single-cell). Nothing seems to really work for the debris that have the same size or are smaller than my cells.
So my question is if someone has another suggestion on something different that I could try that might help.
Thank you!
Filipa Timóteo Ferreira, you can try Ficoll to separate debris. Here is the protocol http://www2.egr.uh.edu/~nvaradar/private/Prots/Fico_LD.pdf.
If you don't have Ficoll, you can try to adjust centrifugation speed-time starting with the lower speed (100g or less up to 400-500g) but longer time (from 2 mins at higher speed up to 10-15mins at lower speed) and inspect the preparation under a microscope. Hope this helps!
Additionally, try passing you cell suspension through a desired size of filter, depending on your cell size. Using filter helped me in getting rid of debris and you would notice more pure population once you do MACS. Good Luck!
Mohammad Aqdas thank you for your help! Indeed I am using different size strainers during dissociation, the problem is that the debris were smaller than my cells. But I tried the Ficoll protocol and it worked very well. I lose some cells but I checked by FACS the suspension that I am able to isolate and it contained all the populations that I am interested in analyzing. So lets hope it works! Thanks again!
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