Cd3 fitc sk7

MA was dried in a glass tube to remove organic solvents and sonicated in a water bath sonicator for 2 hours at 40 °C in 100 μl of a 50 mM citrate buffer pH 4.5 with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (Sigma). While keeping the tube at 37 °C, 20 μg of CD1b monomer was added and incubated overnight at 37°C. The next day, the solution was neutralized to pH 7.4 by adding 1M Tris buffer pH 8.5. Tetramers were formed by adding streptavidin-phycoerythrin or streptavidin-allophycocyanin (Life Technologies). For flow cytometry, cells were stained with tetramer (1:100) for 10 min at room temperature. Subsequently, antibodies were added and incubated for 10 min at room temperature, followed by 10 min on ice. The following antibodies were used: CD3-Fitc SK7 (BD Biosciences), TRAV1-2-PE (Vα7.2) (clone 3C10, Biolegend), CD4-BV421 (Biolegend), CD8-PE (eBioscience), TRBV6-2-Fitc (Vβ13.2) (Beckman Coulter). Analysis was performed on a LSRFortessa (BD Biosciences) equipped with 5 lasers. For all analyses, an initial gate was set based on forward scatter and side scatter to exclude debris and doublets. For TCR sequencing, tetramer-positive populations were sorted on a FACSAria Cell Sorter (BD Biosciences) equipped with 3 lasers.