To investigate the phenotype of neutrophils, freshly isolated NDGs and LDGs were incubated with the following antibodies for 30 minutes on ice: CD10-PerCP-Cy5.5 (clone HI10a, BD Pharmingen, 25μg/mL), CD15-PE-Vio770 (clone VIMC6), CD16-VioGreen (clone REA423), CD33-FITC (clone REA775), CD62L-VioBlue (clone 145/15), CD63-PE (clone REA1055) and CD66b-APC (clone REA306), all Miltenyi Biotec, Germany. Flow cytometry was performed using the MACSQuant® flow cytometer (Miltenyi Biotec, Germany). Data analysis was performed with FlowJo software (TreeStar, CA, version 10.6.1). To determine the absolute numbers of NDGs from peripheral blood, Truecount TM tubes (BD Biosciences) were used according to the manufacturer’s instructions after staining full blood with CD45-FITC (clone REA747), CD16-VioGreen (clone REA423) and CD19-APC (clone REA675, all Miltenyi Biotec) and lysis with immediate fixation using the Fix/Lyse Solution (Invitrogen). In contrast, absolute numbers of LDGs were determined by calculating the percentage of CD15+ LDGs contained in the PBMC-layer from the absolute number of CD45+ PBMCs as determined by Truecount assessment.
Lipka S., Ostendorf L., Schneider U., Hiepe F., Apel F, & Alexander T. (2023). Increased levels of immature and activated low density granulocytes and altered degradation of neutrophil extracellular traps in granulomatosis with polyangiitis. PLOS ONE, 18(3), e0282919.
+ Open protocol