Anti human cd66a c e alexafluor 488

Neutrophils were isolated from peripheral venous blood using a Percol density gradient as described previously^{25 (link)}. A further purification step was performed using the EasySep™ Human Neutrophil Enrichment Kit (STEMCELL Technologies) as per the manufacturer’s instructions^{26 (link)}. Neutrophils were resuspended in Hanks balanced salt solution (HBSS⁻) without calcium and magnesium (Life Technologies) and FACS analysis was performed using Anti-Human CD49d-APC (BioLegend, 304307) and Anti-Human CD66a/c/e AlexaFluor-488 (BioLegend, 342306) antibodies to confirm purity. Prior to migration, neutrophils were stained with a CellTrace Calcein Red-Orange cell stain (Thermo Fisher) as per the manufacturer’s instructions. Stained neutrophils were washed twice in HBSS⁻ then resuspended in HBSS plus calcium and magnesium (HBSS⁺). Unstained neutrophils were used if downstream analysis was to be performed (FACS/ microscopy), these were resuspended in HBSS⁺ prior to migration.

Neutrophils were isolated from peripheral venous blood using a Percol density gradient, as described previously (31 (link)), and were purified using an EasySep human neutrophil enrichment kit (Stemcell Technologies) per the manufacturer’s instructions (32 (link)). Neutrophils were resuspended in Hanks balanced salt solution (HBSS−) (Life Technologies), and fluorescence-activated cell sorter (FACS) analysis was performed using anti-human CD49d-APC (BioLegend, 304307) and anti-human CD66a/c/e Alexa Fluor-488 (BioLegend, 342306) antibodies to confirm purity. Neutrophils were counted using a hemocytometer.