Lightning link r phycoerythrin conjugation kit

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).

Properdin (Calbiochem); human IgG, IgM and IgA (Sigma); EBNA-1 protein (Tebu-Bio) and neutravidin (Thermo Scientific) were coupled to beads and utilized to determine the optimal assay conditions for measurement of complement activation. For detecting the various antibody isotypes, goat anti-human IgM (µ chain specific) F(ab')₂ –Cy3, goat anti-human IgG (γ chain specific) F(ab')₂ –PE or goat anti-human IgA (α chain specific) F(ab')₂ –DyL549 (Jackson ImmunoResearch Laboratories) antibodies were used. Goat anti-human complement C3 specific F(ab')₂ antibody (MyBioSource) was labeled with R-Phycoerythrin (Lightning-Link-R-Phycoerythrin Conjugation Kit, Innova Biosciences) according to manufacturer's protocol and was used to detect complement activation driven C3 deposition on beads.