Cd11b bv785

Total blood counts, leucocyte, and monocyte counts were measured with a Sysmex XN-450 Hematology Analyzer (Sysmex Corporation, Kobe, Japan). Undiluted blood (50 µL) was incubated for 15 min in the dark at room temperature with the following antibodies: CD3-APC-750 (mouse, dilution: 1:25; Beckman Coulter, Woerden, The Netherlands), CD56-APC (mouse, 1:25; Beckman Coulter), CD16-FITC (mouse, 1:25; eBioscience Thermo Fisher Scientific, Breda, The Netherlands), CD14-PC7 (mouse, 1:25; eBioscience Thermo Fisher Scientific), CCR2-BV421 (mouse, 1:50 and 1:25; BD Biosciences), CD36-APC-700 (mouse, 1:10; Beckman Coulter), CD41-PC5.5 (mouse, 1:50; Biolegend, San Diego, CA, USA), CD11b-BV785 (mouse, 1:50 and 1:25; Biolegend). Followed by the addition of 1 mL 10× diluted lysis buffer (BD Pharm Lyse; BD Biosciences), samples were mixed and incubated for 10 min in the dark at room temperature. Samples were then measured on a Beckman Coulter FC500 flow cytometer, each sample was measured both stained and unstained. Flow cytometry data were analysed using Kaluza software (Beckman Coulter); for gating strategy, see Supplementary material online, Figure S1. Besides determining the different monocyte subsets (classical, intermediate, or non-classical). Atherogenic markers CCR2, CD36, CD41, and CD11b were determined per monocyte subset.

For staining with extracellular markers, cells were first treated with Fc block (Biolegend, 101320) in PBS (Fisher, 10010023) with 3% FBS for 30 min. Then, the following antibodies were added: CD3-APC Cy7 (Biolegend, 100221), CD19-APC Cy7 (Biolegend, 115530), CD11c-APC (Biolegend, 117309), CD11b-BV785 (Biolegend, 101243), and live-dead dye (Fisher, L10119), and cells were further incubated on ice in the dark for another 30 min. Cells were then washed with PBS and fixed with 4% paraformaldehyde (PFA) (Fisher, 28908) for 15 min at room temperature. Samples were acquired on a LSR II cytometer (BD) and data were analyzed using FlowJo software.