Hi guys, am working on by flow cytometry analysis of DC and B cells using whole blood assay. Which means i stain , then lyse the RBCs and acquire. However, i couldnt find >400,000 events. I was wondering to acquire 1,000,000 events as the proportion some subsets in my panels is
I have used whole blood in flow before but never had a problem with events(even if most of them are granulocytes). When you say events what gates are you referring to? All cells? PBMCs?
Even with volumes like 3ml I would get 1x10^6 total events easily. The lower end of the normal range for wbc counts is about 4.0x10^5 cells/ml so unless your patients are leukopenic that shouldn't be the problem. However, if you are staining directly in blood this limits you staining volume severely unless you want to burn through antibody. Many people will only use ~100ul of blood which is only 4.0x10^4 to ~1.0x10^5 wbcs in normal people. With high quality antibodies and good titration you can perhaps make this work with larger volumes but determine this empirically.
There are any number of points where you could try to improve cell yeild but the easiest is changing your staining/lysing. If you don't want to increase your staining volume when you use whole blood try lysing rbcs first. I typically stain after RBC lysising blood, this lets you have more control over the number of cells being stained since you can lysis a larger volume of blood and then resuspended at your staining volume. You can even add a cell counting step if you so desire to further standardize your collection. This will require re-titrating your antibodies since your target cell:antibody ratio is shifted significantly from direct blood staining.
Thank you very much William. It helped me a lot. When you say 3ml of blood are you referring whole blood assay for lysis and then stain? And when I say events all events am referring all cells ( granulocytes, Mono, lymph).
Since your describing total events then the issue is the volume of blood used.
By 3ml of blood I mean that's how much blood you lysis for your stain. A basic summary is:
1. Collect Blood in 4ml EDTA Tube(~3.5ml of blood if done with Vacutainer)
2. Invert tube 10x collect 3ml and add to an appropriate volume of RBC lysis buffer, mix and incubate according to your protocol.
3. Wash cells in FACS Buffer 1x
OPTIONAL: Count cells
4. Aspirate wash buffer and re-suspend cells in staining solution, stain as appropriate.
5. Wash 3x in FACS Buffer
6. Run on Flow Cytometer the same day. If you can not run on the same day the fix sample.
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