What is the best method for gating/markers to confirm neutrophils in either:
A)Whole blood
B)Isolated Granulocyte fraction
For whole blood, would it be possible to gate granulocytes by size and then stain for MPO? Neutrophil population should be heavily positive for MPO when compared to other granulocytes and any possible contaminating monocytes?
many thanks.
Hello Corre, I thought Ly6G was only for murine Neutrophils and not human? Either way, Ly6G also has expression on monocytes, so if they contaminate the neutrophil size gate, they could show as positive.
Hi Charlie,
Many of the markers on human neutrophils are shared with monocytes, though often at a different level of expression.
Cytoplasmic MPO is indeed much brighter in neutrophils than monos and other grans. There are people with abnormally low peroxidase, though.
I would still recommend also having CD45 present, and at least some other lineage marker. In mature grans, CD15 or CD16 are nice. CD16 is heavily influenced by maturation, so you'll see left shift easily. Also, eosinophils do not express CD16, but do express slightly higher CD46. So gating on grans by CD45/SSC, you can easily gate out eos in the CD16- area.
Of course, you can end up getting a sample with lots of CD16+ monos. Good to add either CD14 or CD64 for gating on monos.
You can also increase cluster difference with radar graphs if you have access to Kaluza. Hypogranular neutrophils in the mono size/scatter gate will still express CD15 or CD16, plus CD45 differently.
If you have a more specific application of the markers, maybe I can give a more specific recommendation.
Hi Charlie,
For whole blood neutrophil gating, they are positive for: MPO/CD66abce/CD66b/CD11b. HLD-DR is useful in this combination to separate any monocytes contamination. If you want to further separate granulocytes, basophils are positive for CD123. Idk much about eosinophils. There are probably other markers for it. Hope this helps!
Hi,
how permeabilisation affect CD11b-c CD66b staining on neutrophil ?
(whole blood cytometry ?)
THank you .
Human or mouse? For mouse CD45+ /Ly6G+ / CD115- (the later to exclude monocytes) is enough
For human CD45/CD15 gating from SS/FS would enable you to find the population of neutrophils. By looking at the scattergram, you will be able to make differentiation of band neutrophils, eosinophils and mature neutrophils since they have different scatter features.
Hello Charlie Bridgewood ,
You can follow the steps described below.
Step-1 : Treat your sample with ACK lysis buffer
S-2 : Stain with CD45 & Ly6G
S-3: Gate the FSC & SSC high cell populations (a)
S-4: Identify pop. (a) --> CD45 --> Ly6G +ve cells
S-5: Enjoy the delicacy of Neutrophils presence . If you like, sort them out & play along :-D
Best wishes,
Babu
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