Alexa fluor 488 conjugated cd11b 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).

Cell viability, leukocyte activation, and platelet–leukocyte adhesion were studied using the ImageStream^X Mark II imaging flow cytometer (Amnis Corporation) equipped with a 40× objective, 6 imaging channels, and 405, 488, and 642 nm lasers. Whole blood was diluted 1:33 in RPMI 1640 medium (supplemented with 10 mM HEPES; Life Technologies) and stained with the following where applicable: calcein blue, AM (10 µM; Life Technologies); Pacific Blue-conjugated CD41 antibody (1:100; clone HIP8; BioLegend, Cat# 303713); CellEvent Caspase-3/7 Green Detection Reagent (5 µM; Life Technologies); Alexa Fluor 488-conjugated CD11b antibody (1:500; clone ICRF44; Stemcell Technologies, Cat# 60040AD); PE-conjugated CD66b antibody (1:150; clone G10F5; Stemcell Technologies, Cat# 60086PE); PE-conjugated EpCAM antibody (1:250; clone VU1D9; Cell Signaling Technology, Cat# 8995s); PE-CF594-conjugated CD45 antibody (1:666; clone HI30; BD Biosciences, Cat# 562279); and DRAQ5 (1 μM; Cell Signaling Technology). Single cells were gated using the nuclear stain DRAQ5. Viable cells were defined as calcein-positive but caspase-3/7-negative.