Cd16 pe cy5 3g8

PBMC were immunophenotyped using the following antibodies: CD3-PerCP (UCHT1), CD4-APC (OKT4), CD18-PE (TS1/18), CD19-PE (HIB19), NKG2D-APC (1D11), CX3CR1-FITC (2A9-1, all Biolegend), CD3-Pacific Blue (UCHT1), CD8-FITC (HIT8a), CD16-PE-Cy5 (3G8), CD56-PE, -Alexa Fluor 647 or -PE-Cy7 (B159), CD69-FITC (FN50) (all BD Biosciences), CD62L-FITC (LT-TD180) (ImmunoTools), GCR-Alexa Fluor 488 (D8H2) (Cell Signaling Technology) and ADRB2 pure (S364, RayBiotech). The antibody against ADRB2 was coupled to R-phycoerythrin (RPE) in house using the ABSelect BSA Removal Kit to clean and concentrate the pure ADRB2 antibody followed by fluorochrome coupling with the Lightning-Link® R-Phycoerythrin Conjugation Kit according to manufacturer’s instructions (Innova Biosciences).
All stainings were performed as surface-stainings for 30 min. at 4°C, with exception of the intracellular GCR staining that was performed in whole blood according to a standard ICS protocol (Cell Signaling Technology). All sample acquisitions were performed on an Accuri^TM C6 (immune cell subsets and NK function) or a LSRFortessa^TM (NK receptor phenotyping) flow cytometer (BD Biosciences).

IE were stained with PKH26 (Sigma-Aldrich, Castle Hill, NSW, Australia) according to the manufacturer’s instructions. Aliquots of whole blood (50 μL) were incubated with 5 x 10⁶ PKH26-stained CS2-IE for 15 minutes, and processed as above for whole blood phagocytosis except that cells were stained with CD14 Pacific Blue (M5E2, Biolegend) and CD16 PE Cy5 (3G8, BD Biosciences). Samples were acquired using an ImageStream 100 imaging flow cytometer and analysed using IDEAS software.