Pe cy7 conjugated cd16 antibody

Imaging flow cytometry was performed using the ImageStream^X Mark II imaging flow cytometer (Amnis Corporation) equipped with a 40 × objective, 6 imaging channels, and 405 nm, 488 nm, and 642 lasers. For analysis of cell viability and CD45 expression, the enriched leukocytes were resuspended in 0.1% BSA in HEPES-buffered saline after RBC lysis and stained with the following antibodies and stains where applicable: DRAQ5 (1 μM; Cell Signaling Technologies), Sytox Blue (1 μM; Life Technologies), CellEvent Caspase-3/7 Green Detection Reagent (0.75 μM; Life Technologies), FITC-conjugated CD45 antibody (1:500; clone 5B1; Miltenyi Biotec), PE-conjugated CD66b antibody (1:125; clone G10F5; Stemcell Technologies), and PE-Cy7-conjugated CD16 antibody (1:200 or 1:333; clone 3G8; BD Biosciences). Single cells were gated using the nuclear marker DRAQ5. Neutrophils were identified by the dual positivity of CD66b and CD16. For analysis of neutrophil activation post-enrichment, cells were stained with DRAQ5 (1 μM; Cell Signaling Technologies), VioBlue-conjugated CD45 antibody (1:100; clone 5B1; Miltenyi Biotec), Alexa Fluor 488-conjugated CD11b antibody (1:500; clone ICRF44; Stemcell Technologies), PE-conjugated CD66b antibody (1:125; clone G10F5; Stemcell Technologies), and PE-Cy7-conjugated CD16 antibody (1:333; clone 3G8; BD Biosciences).