Fitc antimouse cd86 gl1

All flow cytometric analysis was performed using an LSR II flow cytometer (BD Biosciences) and analyzed with FlowJo version 10.0.7 (Tree Star Inc., Ashland, OR, USA). Antibodies used for flow cytometry: eFluor^® 450 antimouse CD3ε (145-2C11, eBioscience, San Diego, CA, USA), PE-Cy7 antimouse NK1.1 (PK136, BD Pharmingen), PE-Cy7 antimouse CD19 (1-D3, BD Pharmingen), PE antimouse CD4 (GK1.5, TONBO Biosciences, San Diego, CA, USA), FITC antimouse CD8α (53-6.7, BD Pharmingen), APC antimouse IFN-γ (XMG1.2, TONBO Biosciences), Pacific Blue antimouse CD11c (N418, Biolegend, San Diego, CA, USA), FITC antimouse CD86 (GL1, BD Pharmingen), PE antimouse CD40 (3/23, BD Pharmingen), PE antimouse I-A^b (AF6-120.1, BD Pharmingen), PE-Cy5 antimouse CD11b (M1/70, TONBO Biosciences), PE-Cy7 antimouse CD8α (53-6.7, TONBO Biosciences), and APC-Cy7 antimouse CD103 (2E7, Biolegend).

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.