Dear Wei,

I had sorted rare subsets of B cells from rhesus macaques and had this type of issue in the beginning of the procedure. They represented about 0.5% at the best scenario of all PBMCs. Cell purity was highly increased with a simple adjustment in the sorting strategy. Besides checking the nozzle size and diluting your cells a bit more, it helps if you set up split your cell sorting in 2 steps. The 1st step must be performed just to enrich the desired population: Live+ CD-X+. Do not set up the gates for CD3+ and CD4+ cells here! In my case, I had nearly 30% of PBMCs after that procedure instead of 0.3-0.5%. Then, you perform the 2nd cell sorting within that cell subset for the desired cells (CD3+ CD4+). You will see a great improvement in the purity and also will spend less time operating the sorter.

PS - Never used Fcgamma blocking antibodies for macaque cell staining and that was never a problem.

Best of luck,

Eduardo Silveira

Need to know more about the instrument sorting criteria.

Ann Atzberger

Hi thanks for your help.

I'm using either Aria III or II with the nozzle size 70uM.

I resuspended my cells in 1 mL of fixative buffer with final concentration of 3x10^6 cells/mL.

I sorted my cells with 70uM nozzle size (drop 1 about 150 with Gaps about 6) in 4 way purity and 1300events/sec.

I will provide all information you looking for, dont hesitate to ask me please.

Thanks.

P.S. this marker X is suspected as kind of activation marker.

Eduardo L. V. Silveira

Thanks for your information !!!

I have tried this method as you suggested.

First, I sorted the most desired population out Live+ CD-X+ (up to 97.7%).

Unfortunately, most CD3+ CD4+ cells population in first step sorted population (including Live+ CD-X+ 97.7 & Live+ CD-X- 2%) are CD-X - up to 80%.

Therefore, I reconsidered the 2nd step sorting strategy and I sorted CD3+ CD4+ CD X + (sort of 2 step purification based on CD X + ) then. In final sorted population, I could achieved the 70% purity of CD3+CD4+ CDx + cells with acceptable cell No.

I took some suggestions from everyone.

1st: I used 85uM nozzle size instead of 70uM because of CD X is a suspected activation marker.

2nd: I double the amount of CD X+ antibody as my cell number No. is about 5 folds higher than that I used for immunophenotyping analysis.

By the way, I am wondering if my strategy for sorting is correct.

Because I apply CD X + based sorting on both 1 st step & 2nd step sorting.

Did you calculate the drop delay before sorting? The 70um nozzle is probably ok since the cells are fixed. What is the actual setting for the Flow Rate, ..I do not see a dead cell stain and it could be that the positive population is non specific staining of dead cells. You can try a live/dead fixable stain.

You say the cells are activated, hence expect an increase in size: make sure you are not excluding them in the FSC/SSC gate: does your CD-X antibody specifically identify the population; if yes plot the FSC against CD-X on total cells to identify where these cells are; gate the positive population and back-gate this to the FSC/SSC plot. You might need to add in another marker to exclude monocytes.