Clone 63d3

Whole human blood was incubated in ACK RBC lysis buffer (Thermo Fisher Scientific) for 4 min at 37 °C to remove red blood cells. Cells were incubated with NIR Zombie (1:200, Biolegend) in PBS for live/dead staining for 15 min at room temperature, followed by BV421 CD45 (1:200, clone 2D1, #368522, Biolegend), APC CD14 (1:200, clone 63D3, #367118, Biolegend), BV605 CD16b (1:200, clone CLB-gran 11.5, #735143 BD), PE/Cy7 CD19 (1:200, clone HIB19, #302216, Biolegend), PE/Cy7 CD3 (1:200, clone HIT3a, #300316, Biolegend) and Fc blocker (1:100, anti-mouse CD16/32,BD) at 4 °C in the dark for 20 min, and washed in PBS/0.5% BSA. Neutrophils, lymphocytes, and monocytes were identified from peripheral blood cells of normal healthy donors with the following high dimensional immunophenotyping; Neutrophils (CD45⁺, CD3/CD19−, CD16b+, CD14⁻), lymphocytes (CD45⁺, CD3/CD19+, CD16b−, CD14⁻) and monocytes (CD45⁺, CD3/CD19−, CD16b−, CD14⁻). Analysis was performed using an LSRII (BD) flow cytometer and FlowJo. v10 (Treestar).

PBMCs were freshly isolated as previously described (Cooper et al., 2008 (link)) from healthy volunteers using a double density gradient of histopaque (Sigma-Aldrich). PBMCs were pre-incubated with apoA1 for 45 min at 37°C, diluted to 1 × 10⁶ cells/ml in PBS-BSA (5%) and stimulated with C5a (10 nM) for 5 min and then stained with active CD11b (Clone CBRM 1/5; eBioscience), total CD11b (Clone ICRF44; Biolegend), CD49b (Clone P1E6-C5; Biolegend), CD14 (Clone 63D3; Biolegend), C5aR1 (Clone S5/1; Biolegend) and CD62L (Clone DREG-56; Biolegend) and analysed by flow cytometry.