I am analyzing bronchoalveolar lavage fluids from infected mice to evaluate leukocyte infiltration with flow cytometry. I am staining BALF cells with anti CD45 (pan leukocytes), CD11b (myeloid cells), Ly6G (PMN), CD3 (lymphocytes), and F4/80 (macrophages).
I see two, very distinct macrophage populations: one that is F4/80 bright CD11b+ and one CD11b bright but F4/80 negative and have lower FSC and SSC intensity.
I thought the F4/80 bright/CD11b+ are the resident alveolar macrophages, while the second population represents macrophages derived from monocytes (that haven't acquired F4/80 yet). Am I correct? Is there any additional marker/criteria I can use to distinguish these two cell subsets?
I Dominic.
Thanks for your answer. I am confident the F4/80 positive cells are not eosinophils, based on their FSC/SSC profile. I will check if they are DC using CD11c. Like yoy said, I see lots of CD11b+ under inflammatory conditions, some are Ly6G+ PMN, some are F4/80+, some F4/80 negative. I can have up to 8 colors in my FC panel, so I will include other markers. THanks for the papers.
Antonio
Dominic gave a pretty good answer..
autofluorescence (in FITC although macs are also autofl in PE, and pacific blue) would be another criteria to include. and if your focus is only on macrophages I would recommend put all the other lineage markers in the same dump channel.. and since in an inflammatory condition sometimes its difficult to differentiate I would suggest do a FACS sort of the populations you gate as macs and a cytospin on the sorted cells to look into their morphology ..
Hi, I agree with Dominic, macrophages in the lung are complicated, particularly as they match many of the same markers as DCs and inflammatory DCs and overlap some in SSC/FSC with activated eosinophils . DCs will not express Siglec-F, but eosinophils and alveolar macrophage will. If you can distinguish eosinophils with CCR3 or Gr1, then you can use Siglec-F for alveolar macrophage to distinguish from DCs as well.
Here are another two articles that show how complicated DC vs macrophage are in the lung.
http://www.jci.org/articles/view/39717
http://www.annualreviews.org/doi/pdf/10.1146/annurev-immunol-020711-075021
Of note:
Eosinophils in the lung are identified as F4/80lmed, CCR3+, Siglec-F+, IL-5Ra+ (faint marker in mice), Gr1med/lo (spans a wide range, but is never as high as neutrophils), Cd11c- Cd11b+ .
Good luck.
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