Quantification of Blood Cell Surface Receptors

For baseline quantification of surface receptors, blood leukocytes were stained and analyzed using flow cytometry as previously described [16 (link)]. Whole blood samples were collected into K₂EDTA vacutainers (BD Biosciences) during the same blood draw described above. Samples were centrifuged at 400x g for 10 min at 4°C, plasma was removed, and cells were resuspended to the original volume in ice cold PBS containing 2.5 mM EDTA. Cells were kept on ice and in the dark for the remaining steps. Cells were incubated with a live/dead discriminator (Life Technologies) and an Fc-blocking reagent (TruStain FcX, BioLegend) for 15 min before an antibody cocktail was added for an additional 15 min. The antibody cocktail consisted of antibodies against CD16 (clone 3G8, BioLegend), CD14 (clone 61D3, eBioscience), CXCR2 (clone 5E8-C7-F10, eBioscience), activated CD11b (clone CBRM1/5, eBioscience), CD63 (clone H5C6, eBioscience), and CD66b (clone G10F5, eBioscience). Live, singlet cells were analyzed using a LSR Fortessa flow cytometer (BD Biosciences) and gated based on forward scatter, side scatter, and surface marker expression (neutrophils are defined as CD16^High FSC^High SSC^Mid, monocytes as CD14^High CD63^High FSC^Mid SSC^Mid, and NK cells as CD16^Mid FSC^Low SSC^Low).