Multicolor Flow Cytometry Protocol for Immune Cell Profiling

Leukocytes were stained with the following antibodies: F4/80 (BM8)-PEcy7 (BioLegend), Ly6C (AL-21)-APCCy7, Ly6G (1A-8)-APC, CD11b (M1/70)-BV421, CD206 (19.2)-APC, and CD3 (SK7)-BV421 (BD Biosciences). Total macrophages (F4/80⁺), monocytes (Ly6C⁺F4/80^–), neutrophils (Ly6G⁺), and lymphocytes (CD3⁺) were evaluated. Events were acquired in FACSCanto II (BD Biosciences) and analyzed using FlowJo Software (Tree Star Inc.). Macrophage phenotypes were defined by the expression of F4/80, CD11b, Ly6C, and CD206, as described previously (16 (link), 25 (link)).

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Zaidan I., Tavares L.P., Sugimoto M.A., Lima K.M., Negreiros-Lima G.L., Teixeira L.C., Miranda T.C., Valiate B.V., Cramer A., Vago J.P., Campolina-Silva G.H., Souza J.A., Grossi L.C., Pinho V., Campagnole-Santos M.J., Santos R.A., Teixeira M.M., Galvão I, & Sousa L.P. (2022). Angiotensin-(1-7)/MasR axis promotes migration of monocytes/macrophages with a regulatory phenotype to perform phagocytosis and efferocytosis. JCI Insight, 7(1), e147819.

Publication 2022

F4/80 (BM8)-PEcy7 FACSCanto II

Antibodies Cd11b Leukocytes Lymphocytes Macrophage Monocytes Neutrophils Phenotypes Tree

Corresponding Organization : Universidade Federal de Minas Gerais

Other organizations : Brigham and Women's Hospital

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Variable analysis

independent variables

Antibodies used for staining leukocytes: F4/80 (BM8)-PEcy7 (BioLegend), Ly6C (AL-21)-APCCy7, Ly6G (1A-8)-APC, CD11b (M1/70)-BV421, CD206 (19.2)-APC, and CD3 (SK7)-BV421 (BD Biosciences)

dependent variables

Total macrophages (F4/80+)
Monocytes (Ly6C+F4/80–)
Neutrophils (Ly6G+)
Lymphocytes (CD3+)

control variables

Events were acquired in FACSCanto II (BD Biosciences) and analyzed using FlowJo Software (Tree Star Inc.)
Macrophage phenotypes were defined by the expression of F4/80, CD11b, Ly6C, and CD206, as described previously (16, 25)

controls

No positive or negative controls were explicitly mentioned in the information provided.

Annotations

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