Hi there,
I am attempting to isolate canine lymphocytes with MACS positive selection. I am planning to first use Ficoll to isolate PBMCs, then use MACS to sort the lymphocyte population, and flow cytometry to assess purity. My Ficoll isolation protocol has so far had neutrophil contamination up to 20%, so I was hoping to use two rounds of MACS sorting to get rid of neutrophil contamination (first with CD3, then with CD4 to separate the T lymphocyte population further). I am using CD3 first since canine neutrophils express CD4.
My question are:
1. Is performing two rounds of MACS positive selection feasible? Will it affect cell viability in future studies?
2. Will two rounds of positive selection affect flow cytometry results?
Any help would be greatly appreciated.
1. Two rounds of MACS enrichment is routinely performed to increase the purity. In fact, AutoMACS has a program to perform two rounds of enrichment. I do find around 5% decreases in viability after two rounds of MACS.
2. I don't see any reason that two rounds of MACS compared to one round affect FACS results.
1. Yes it is feasible. Cell viability slightly affected depending on your working time and manipulation of cells.
2. Facs results will show more enrichment for the population of interest.
3. What not after first CD3 enrichment do sorting cells by population for high purity (for sequencing as Macs will never give you more then 85-90% max purity + you don't have to do facs afterwards).
Good luck.
Maybe you can also get rid of the contaminating neutrophils by an additional depletion step? If you have an biotinylated antibody against canine neutrophils you could load it into Strep-Tactin® TACS Agrose columns (https://www.iba-lifesciences.com/strep-tactin-tacs-agarose-column/6-6310-001). Just just have to pass your sample through this column and get rid of the neutrophils. This way you won't need an extra step involving magnetic beads. I attached an application note that shows this option of cell depletion (it's for human cells, but it should be easily adaptable for canine cells).