αcd3 fitc

For all experiments, frozen PBMCs were thawed and rested for 16-18h in complete RPMI 1640 media at 37°C in a 5% CO₂ incubator before starting the experiments. Phenotypic analyses of PBMCs were performed using surface antibody staining for 30 minutes at 4°C, using combinations of the following anti-human monoclonal antibodies: αCD3-PE/Dazzle594 (OKT3), αCD3-FITC (OKT3), αCD38-PE/Cy7 (HB-7), αCD19-BV650 (HIB19), αCD56-BV750 (5.1H11), αCD4-PerCP/Cy5.5 (OKT4), αCD8-AF700 (SK1), αCCR7-APC/Fire (G043H7), αCD45RO-BV711 (UCHL1), all purchased from BioLegend. After staining the cells with antibodies for the phenotypic markers cells were fixed in 1% paraformaldehyde and acquired in a flow cytometer. The functional capacity of T cells was determined by analyzing the intracellular cytokine production upon stimulation with overlapping HIV-Gag polyprotein peptides using αIFNγ-APC (4S.B3) and αTNFα-PE (MAb11) antibodies from BioLegend. After staining the cells for surface markers, cells were permeabilized with Cytofix/Cytoperm (BD Biosciences) followed by intra cellular cytokine staining and finally fixed in 1% paraformaldehyde. Live/dead cell discrimination was performed using Zombie Aqua fixable viability Kit (BioLegend). Data were acquired and compensated using Cytek Aurora (Cytek Biosciences) flow cytometer and further analyzed with FlowJo (version 10.7.1).

Antibodies were diluted 1:100 on 1 × 10⁶ cells for flow cytometry; Cytotect (Biotest); αCD107a-APC (BD FastImmune clone H4A3); αCD56-BV650 (Biolegend clone 5.1H11); αCD3-FITC (Biolegend clone UCHT1); αhuman-IgG-PE (BD); human Fcγ-TexasRed (Rockland); rituximab (Rtx, Roche); herceptin (Hc, Roche); αhuman-IgG-PE (Miltenyi Biotec); αHis-PE (Miltenyi Biotec); αCD20-PE (Miltenyi Biotec); polyclonal rabbit-αhuman IgG-FITC (ThermoFisher); THE Anti-His-HRP (Genscript); MSL-109 anti HCMV gH (Absolute Antibody).

Phenotypes of PBMCs were analyzed using antibody staining for 30 min at 4 °C and flow cytometry acquisition (BD LSRII; BD Biosciences) with combinations of the following anti-human monoclonal antibodies: αCD3-AF700 (UCHT1), αCD3-FITC (OKT3), αCD4-PE/Cy7 (OKT4), αCD4-BV421 (OKT4), αCD8-BV510 (SK1), αCCR7-FITC (G043H7), αCD19-BV510 (HIB19), αCD21- PE/Cy7 (Bu32), αCD27-APC (M-T217), αPD-1-BV421 (EH12.2H7), αCD244(2B4)-PerCP/Cy5.5 (C1.7), αPD-L1-PE (MIH2) all purchased from BioLegend and αCD45RO-APC-eFluor780 (UCHL1) from eBiosciences. Cytokine production from T cells upon stimulation with overlapping peptides of HBV was used to assess their functional capacity. Production of IFNγ, TNFα and IL-2 was measured by intracellular staining with αIFNγ-APC (4S.B3), αTNFα-PE (MAb11) and αIL-2-PerCP/Cy5.5 (MQ1-17H12) from BioLegend. Live/dead cell discrimination was performed using Zombie NIR fixable viability Kit (BioLegend). Cells were permeabilized with Cytofix/Cytoperm (BD Biosciences) and fixed in 1% paraformaldehyde. Data were acquired and compensated using FACS Diva software (BD Biosciences) and analyzed with FlowJo (version 10).