I tried isolating monocytes from human PBMCs using positive selection (CD14 positive magnetic beads). When I stained my isolated cells to confirm with flow cytometry, I got a big proportion of CD15-bright CD14dim cells suggesting granulocyte isolation as well. Haven't stained for live/dead cells. The markers I used for staining were CD14, CD15 (to exclude granulocytes) and CD45 (for leukocytes), expecting to get a big population of CD14(+) CD45 (+) CD15 (-) cells.
If you are using PBMCs, I don't see how you can have many neutrophils, since that would have been below the PBMC layer.
Silly question... are you sure your CD14 isn't blocked by the positive selection?
Second, the most obvious is that your positive gate for you CD15 is wrong...
What exactly is your panel design?
Thank you for your answer Shen-An Hwang. I use the CD14 antibody suggested by company as control for positive CD14 selection. I also stain for CD15 to exclude granulocytes and CD45 for leukocytes.
So that's the thing... you don't need a marker to exclude granulocytes, you can do that using FSC and SSC. Although with ficoll, I still don't see how you have that much contamination.
If you can put up your flow plots the community might be able to help you pinpoint the issues.
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining