Anti igd bv 605

Cryopreserved PBMCs were centrifuged and suspended in PEB buffer (PBS with 0.5% BSA and 2 mM EDTA) and incubated with Fc receptor block (Miltenyi, 130-059-901) for 15 min at 4 °C (dilution 1:10). Next, cells were washed in PEB and stained for 30 min in brilliant stain buffer at 4 °C in the dark using the following antibodies together with both the PE-and APC-conjugated recombinant RBD tetramers: anti-CD3-APC Fire 810 (BioLegend, 344858; diluted 1:100); anti-CD11c-BV785 (Bio-Legend, 301644; 1:50); anti-CD19-PE Vio770 (Miltenyi, 130-113-170; 1:100); anti-CD20-BV421 (BD, 562873; 1:100); anti-CD21-BUV496 (BD, 750614; 1:50); anti-CD27-PercP-Vio700 (Miltenyi, 130-113-632; 1:100); anti-CD38-Viobright FITC (Miltenyi, 130-113-433: 1:50); anti-IgM-PE-CF594 (BD, 562539; 1:50); anti-IgD-BV605 (BioLegend, 348232; 1:50); and anti-IgG-BV480 (BD, 746341; 1:50). Cells were washed in PEB and resuspended in a PEB dilution (1:500) of the fixable viability dye eFluor 780 (eBiosciences, 65-0865-18). They were next washed and fixed with 4% paraformaldehyde for 20 min at 4 °C in the dark before a final wash and resuspension for analysis. Cells were then acquired on a Cytek Aurora spectral flow cytometer equipped with five lasers operating at 355 nm, 405 nm, 488 nm, 561 nm and 640 nm using the Spectro-Flo V.2.2.0 (Cytek) software. Data were analysed using FlowJo 10.6.1 software (BD).