Anti cd19 fitc clone hib19

PBMCs were stained with antibodies anti-CD19 FITC (Clone HIB19, Cat No.: 302206, BioLegend, SanDiego, CA, USA) and anti-Galectin-3 PE (Clone B2C10, Cat No.: 565676, BD Biosciences, Franklin Lakes, NJ, USA). B lymphocytes with Gal-3 membrane expression were analyzed within CD19+ cells (Figure 1A). For intracellular assessment of Gal-3 expression, the Fc receptor was blocked (FcR Blocking Reagent, Cat No. 130-059-901, Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer instructions. Then, lymphocytes were fixed and permeabilized with Cytofix/Cytoperm Fixation/Permeabilization Kit (Cat No. 554714, BD Biosciences, Franklin Lakes, NJ, USA). Next, cells were stained with monoclonal antibodies against Gal-3 and incubated for one hour in darkness. The acquisition of all probes was performed on CytoFlex LX (Beckman Coulter, Brea, CA, USA), and then cytometric data were analyzed with Kaluza Analysis (Beckman Coulter, CA, USA).

Cryopreserved PBMCs were thawed at 37°C and resuspended in RPMI 10% FBS, washed with PBS 1X and fixed with 1% paraformaldehyde for 20 min at room temperature. After washed with FACS solution (PBS 1%, sodium azide 0.05% and 2% FBS), cells were incubated for 30 min at 4°C with the following antibody cocktail: anti-CD3 PerCP/Cy5.5 (clone SKY; Biolegend; 344808), anti-CD19 FITC (clone HIB19; Biolegend; 302206), anti-CD20 PE Cy7 (clone 2H7; Biolegend; 302312), anti-CD27 APC (clone O323; Biolegend;356410), and anti-CD38 PE (clone HIT2; Biolegend; 980302). Flow cytometry analysis was performed in a FACS Aria II (BD Biosciences, San Jose, CA, USA). Doublets and CD3+ events were gated out. Plasmablasts were defined as CD3⁻/CD19⁺/CD20⁻/CD27+/CD38⁺. 500-1000 plasmablasts were acquired per sample. Analysis was performed using Flowjo software (TreeStar).