Flow cytometry assays on scavenger receptors
Cell surface expression of scavenger receptors, SR-A1 and CD36, were quantified on peritoneal macrophages from female mice by flow cytometry after immunostaining with fluorochrome conjugated antibodies. Fluorescence intensity was quantified on a FACSCalibur flow cytometry instrument with FlowJo software (BD Biosciences). >10,000 total events were acquired to obtain adequate macrophages numbers. The following antibodies were used to stain macrophages: CD36 mAb FITC (Cayman Chemical), anti-mouse SR-AI/MSRA1 (R&D Systems), goat anti-rat IgG-FITC (Santa Cruz Biotechnology), Alexa Fluor® 647 anti-mouse F4/80 (eBioscience), Alexa Fluor® 647 anti-mouse CD11b (eBioscience) and the isotype controls, Alexa Fluor® 647 rat IgG2b (eBioscience), Alexa Fluor® 647 rat IgG2a (eBioscience), normal mouse IgA-FITC (Santa Cruz). Cells were incubated with antibodies for 30 min at 4°C and washed with 0.1% BSA in PBS. Cells with double positive staining for F4/80 and CD11b were gated as macrophage49–51 for the quantification of fluorescence intensity for CD36 and SR-A1 (Supplementary Figure 20), with results normalized to F4/80.