Lsr 2 cytofluorometer

Flow cytometric analysis was performed as previously described [19 (link)]. Briefly, harvested BMDM were incubated in 1 μg/mL of anti-mouse Fc receptor antibody in 100 mL PBS containing 0.5% BSA plus 0.02% NaN₃ (FACS buffer) for 15 min on ice. Subsequently, single-cell suspensions were stained for 15 min at 4°C with blue-fluorescent reactive dye, L23105 (Life Technologies) to discriminate dead cells. After washing, 1-3 × 10⁶ cells were surface-stained in FACS buffer for 15 min at 4°C with antibodies recognizing CD11b (Alexa Fluor 700, BioLegend), F4/80 (Brilliant Violet 785, BioLegend), CD86 (Brilliant Violet 421, BioLegend), PD-L1 (PE-Cy7, BioLegend), and PD-L2 (PE, BioLegend). Surface-stained cells were washed three times with FACS buffer and treated with Fix/Perm reagent according to the protocol of the cytofix/cytoperm kit (BD Biosciences, San Jose, CA, USA). The cells were intracellularly stained in FACS buffer containing anti-Nos2 (PE, eBiosciences) and anti-h/m arginase 1 (APC, R&D systems) for 30 min at 4°C and further collected on an LSR II cytofluorometer (BD, Franklin Lakes, NJ). Stained cells were gated according to size (SSC-A) and forward scatter (FSC-A) to eliminate debris. Doublets were excluded from the analysis by using forward scatter height (FSC-H) and FSC-A. Data analysis was performed using FlowJo Software (FlowJo, LLC).

We isolated the abdominal aortic cells as described previously^{15 (link)}. Harvested cells were counted per mouse and incubated in saturated doses of anti-mouse Fc receptor in 100 μl of ice-cold FACS buffer (1% bovine serum albumin/0.01% NaN₃ in PBS) for 15 min. After washing, 2 × 10⁶ cells were stained with various combinations of antibodies in ice-cold FACS buffer for 15 min, and further collected on a LSR II cytofluorometer (BD Biosciences). Gating strategies are shown in Supplementary Fig. 8. Data were analyzed with FlowJo software (Tree Star).