Human M2 macrophages and murine peritoneal exudate cells were suspended in FACS buffer (DPBS with 1% bovine serum albumin and 0.1% sodium azide) and incubated with Fc block (15 min, 4°C; BD PharMingen). Human M2 macrophages were incubated with anti-human fluorescein isothiocyanate (FITC) CD206 (clone 19.2) (BD Biosciences) and anti-human PerCP/Cy5.5 CD163 (clone RM3/1) (BioLegend) for phenotyping or intracellular staining with anti-human PE HIF-1α (clone 546-16, BioLegend). Murine peritoneal exudates were incubated with anti-mouse PerCP/Cy5.5 CD45 (clone 30-F11 BioLegend, CA, USA), anti-mouse PE/Cy7 CD11b (clone M1/70, eBioscience), anti-mouse APC F4/80 (clone BM8, eBioscience), anti-mouse FITC Ly6C (clone HK1.4, BioLegend), and anti-mouse PE Ly6G (clone 1A8, BioLegend) or appropriate isotype controls. Murine ex vivo efferocytosis was measured by surface labeling with anti-mouse APC F4/80 (clone BM8, eBioscience), followed by intracellular staining of neutrophils with anti-mouse FITC Ly6G (clone 1A8, BioLegend) and RBC with anti-mouse PE TER119 (clone TER-119, BioLegend) or isotypes. All flow cytometric samples were assessed using FACSDiva Canto II (BD Biosciences) and analyzed using FlowJo version X (TreeStar) and using procedures reported in (20 (link)).
Protocol detail
Phenotyping and Efferocytosis Analysis of Macrophages
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Bovine serum albumin
Buffer
Cd11b
Cd163
Cells
Clone
Cy5 5
Flow cytometric
Fluorescein
Human
Intracellular
Isothiocyanate
Isotypes
Macrophages
Mouse
Neutrophils
Peritoneal exudates
Sodium azide
Corresponding Organization : Harvard University
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Variable analysis
independent variables
- Human M2 macrophages
- Murine peritoneal exudate cells
- Phenotyping of human M2 macrophages (CD206, CD163, HIF-1α)
- Phenotyping of murine peritoneal exudate cells (CD45, CD11b, F4/80, Ly6C, Ly6G)
- Murine ex vivo efferocytosis (F4/80, Ly6G, TER119)
- FACS buffer (DPBS with 1% bovine serum albumin and 0.1% sodium azide)
- Fc block (15 min, 4°C; BD PharMingen)
- Appropriate isotype controls
- Positive control: Not explicitly mentioned.
- Negative control: Appropriate isotype controls.
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