Anti cd3 fitc clone ucht1

Human DMCs and PBMCs were incubated with fluorescein-conjugated anti-human monoclonal antibodies (mAbs) including anti-CD3 FITC (clone: UCHT1; BD Biosciences, USA), anti-CD4 V500 (RPA-T4; BD Biosciences, USA), and anti-CD8a PerCP/Cy5.5 (HIT8a; BioLegend, USA) in 1 mL PBS containing 3% (v/v) fetal bovine serum (FBS) at 4°C for 30 min. Paired dCD8 and pCD8 T cells were isolated from the DMCs and PBMCs, respectively, by sorting on an FACSAria III (BD Biosciences, USA) based on the surface expression of CD3, CD4, and CD8 (CD3⁺CD8⁺CD4⁻) with a purity always greater than 95%.

Anti-CD3-FITC (clone UCHT1) and anti-CD69-APC (clone FN50) were both purchased from BD Biosciences (San Jose, CA, USA). Unloaded human CD1d tetramers, and CD1d tetramers loaded with the alpha-galactosylceramide analog PBS57 [17 (link)] and complexed with PE-conjugated streptavidin, were provided by the National Institute of Allergy and Infectious Diseases tetramer facility (Emory University Vaccine Center, Atlanta, GA, USA). PBMCs were isolated from the whole blood of control and AGS subjects using a Ficoll–Paque gradient and stained with fluorescently labeled antibodies against CD3 and CD69 in flow cytometry staining buffer (phosphate buffered saline, 2% fetal calf serum, 0.02% NaN₃). Before fixation and after monoclonal antibody staining, cells were stained with tetramer for 30 min at 4 °C, as described by Kamath et al. [64 (link)]. Samples were acquired on a CyAN ADP flow cytometer (Beckman Coulter, Brea, CA, USA) or on an Attune NxT flow cytometer (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed using FlowJo v10 software (FlowJo LLC, Ashland, OR, USA).