Anti cd3 clone sk7 fitc

PBMC were obtained from leukoreduction collars provided by the Brigham and Women’s Hospital Specimen Bank, as approved by the Partners Healthcare IRB. Human PBMC and PBMC derived T-cells were stained with tetramers at 2 μg ml⁻¹ in PBS containing 1% BSA and 0.01% sodium azide. Cells and tetramer were incubated for 10 min at room temperature, followed by addition of 3 μl of anti-CD3 (clone SK7-Fitc from BD biosciences) per 50 μl staining and another incubation for 10 min at room temperature and 20 min at 4 °C. PBMC were sorted for positive staining with anti-CD3 and the indicated tetramers (Supplementary Fig. 1–5). Expansion of sorted cells was performed using anti-CD3 at 30 ng ml⁻¹ (clone OKT3), irradiated feeder cells, and IL-2. After 2 weeks, the sorting and expansion procedure was repeated once or multiple times. When the resulting cell lines consisted mainly of tetramer⁺ T-cells, they were screened for binding to a panel of antibodies against TCR Vβ and 2 μl of anti-CD4 (clone RPA-T4 from Biolegend) per 50 μl staining. Suitable combinations of antibodies were used to isolate subsets of the original T-cell lines (Supplementary Fig. 1-5). IFN-γ ELISPOT was performed using the 1D1K and GB-11-biotin antibodies (Mabtech), according to the manufacturer’s instructions.

CD1 monomers were obtained from the NIH tetramer facility. For loading of monomers, 32 μg of lipid was sonicated at 37 °C for 2 h in 90 μl of 0.5% CHAPS 50 mM sodium citrate buffer pH 7.4 in 10 mm diameter glass tubes. Subsequently, 20 μg CD1b monomer was added to the tubes and incubated overnight at 37 °C. Molecular Probes streptavidin-APC or streptavidin-PE was used for tetramerization. Human PBMC and T-cell lines were stained with tetramers at 2 μg ml⁻¹ in PBS containing 1% BSA and 0.01% sodium azide. Cells and tetramer were incubated for 10 min at room temperature, followed by addition of 3 μl of anti-CD3 (clone SK7-Fitc from BD biosciences) per 50 μl staining and another incubation for 10 min at room temperature and 20 min at 4 °C. Cells were analyzed using the BD LSRFortessa flow cytometer and FlowJo software.