Igd fitc

Cells from heparinized blood were phenotypically analyzed in a 10-color MoAb conjugate combination using a Navios™ instrument with 10-color PMTs and three solid-state lasers (Beckman Coulter, Fullerton, FL). The list mode data files were further analyzed using Kaluza™ software (Beckman Coulter). In order to guarantee that the optics, laser, fluidics and fluorescence intensity were stable during all measurements calibration was performed using Flow Check Pro Fluorospheres (Beckman Coulter) and Cyto-Cal Multifluor + Violet Fluorescence Alignment Beads (Thermo Scientific, Fremont, CA, USA). After erythrocyte lysis (BD Pharm-Lyse, BectonDickinson) cells were washed with PBS with 1 % bovine serum albumin before being labeled with fluorochrome-conjugated mAbs. After incubation for 30 minutes at 4 °C in the dark, cells were washed twice to remove unbound antibodies and analyzed. For cell surface staining, the following mAbs were used: IgD-FITC, IgM-PE (both Dako, Denmark) and CD3-ECD, CD4-PECy5.5, CD27-PECy7, CD20-PacB, CD45-KromeOrange, CD56-APC, CD8-APC-Alexa Fluor700 and CD19-APC-Alexa Fluor750 (all Beckman Coulter, Marseille, France). Subsequently, the various lymphocyte subpopulations were analyzed on the flow cytometer using CD45/SSC to gate the lymphocyte population.

BA.4/5 S-specific or BA.4+all S-specific single B cell sorting was performed as previously described^{7 (link),26 (link)}. Briefly, PBMC were stained with LIVE/DEAD Fixable Aqua dye (Invitrogen) followed by recombinant trimeric S-twin-Strep of BA.4/5 or BA.4+all. Cells were then incubated with CD3-FITC (1:10 dilution, BD, Cat. 555332), CD14-FITC (1:10 dilution, BD, Cat 555397), CD16-FITC (1:10 dilution, BD, 555406), CD56-FITC (1:50 dilution, BD, Cat. 562793), IgM-FITC (1:10 dilution, BD, Cat. 555782), IgA-FITC (1:50 dilution, Dako, Cat. F0188), IgD-FITC (1:10 dilution, Dako, Cat. F0189), IgG-BV786 (1:20 dilution, BD, Cat. 564230) and CD19-BUV395 (1:50 dilution, BD, Cat. 563549), along with Strep-MABS-DY549 (1:50 dilution, iba, Cat. 2-1566-050) to stain the twin strep tag of the S protein. IgG+ memory B cells were gated as CD19+, IgG+, CD3−, CD14−, CD56−, CD16−, IgM−, IgA− and IgD−, and S+ were further selected, and single cells were sorted into 96-well PCR plates with 10 µl of catching buffer (Tris, Nuclease free-H₂O and RNase inhibitor). Plates were briefly centrifuged at 2000×g for 1 min and left on dry ice before being stored at −80 °C.