Pe anti mouse human cd11b m1 70

Fixed single-cell suspensions were washed twice with 0.1 M glycine in PBS to neutralize excess PFA. Cells were blocked with CD16/32 antibodies (BioLegend Inc, San Diego, CA) in FACS staining buffer (5% FBS, 1% BSA, 0.2% Gelatin) or Cell staining buffer (BioLegend Inc, Cat # 420201) for 15 min to 1 hr. Infected cells were identified by staining them with Alexa Fluor 647 or Alexa Fluor 488 conjugated antibodies to Glycogag (mAb34 hybridoma). Hematopoietic cell types from footpads were identified using APC Rat anti-mouse CD45 (30-F11) (BD-Pharmingen, Cat # 559864), B cells were identified using PE/Cy7 anti-mouse CD19(6D5) (BioLegend, Cat # 115519, RRID:AB_313654), CD4⁺ T cells were identified using PE/Cy7 anti-mouse CD4 (GK1.5) (BioLegend, Cat # 100421, RRID:AB_312706) and APC anti-mouse CD3ε Antibody (145-2 C11) (Cat # 100312, RRID:AB_312677), DCs were identified using AF647 anti-mouse CD11c (N418) (BioLegend, Cat # 117314, RRID:AB_492850) and PE anti-mouse/human CD11b (M1/70) (BioLegend, Cat # 101208, RRID:AB_312791). The cells were incubated with antibodies for 1–2 hr at room temperature. Flow cytometry was performed on a Becton Dickinson Accuri C6 benchtop cytometer. Data were analyzed with Accuri C6 or FlowJo v10 software (Treestar, Ashland, OR). 200,000–500,000 viable cells were acquired for each sample.

For characterization of differentiated and polarized macrophages, dead cells were first excluded by live/dead staining (Ghost Dye Violet 510 Viability Dye, Cell Signaling Technology) and single cells were gated by plotting the height against the area of FSC. Cells were single stained with the following antibodies: PE anti-mouse/human CD11b (M1/70; BioLegend), APC anti-human CD163 (GHI/61; BioLegend), FITC anti-human CD197 (G043H7; BioLegend), FITC anti-human CD206 (15–2; BioLegend), PE anti-human CD36 (5–271, BioLegend), FITC anti-mouse CD86 (GL1; BD), Brilliant Violet 421 anti-mouse CD206 (C068C2, BioLegend). The percentage of positive cells and the median of fluorescence intensity were quantified by flow cytometry using FACS Canto II (BD). A minimum of 30,000 cells was assessed. Data were acquired and analyzed using FACSDiva software (BD).
To measure the incorporation Dil-labeled oxidized LDL (oxLDL) by M1- and M2a-like macrophages, cells (1–2 × 10⁵) were cultured for 24 h in HBSS supplemented with 0.3% bovine serum albumin in the presence or absence of 1000 ng/ml NETs. Next, cells were incubated for 6 h with 20 μg/ml Dil-oxLDL (Thermo Scientific) at 37°C, washed twice with PBS and resuspended in 400 μl PBS + 2% FCS+ 1 mM EDTA for immediate FACS analysis.