Anti cd8a apc clone 53 6

To obtain streptavidin-PE-conjugated tetramer, biotinylated D^d/NP_166–174 monomeric protein was purified and tetramerized at an 8:1 molar ratio with streptavidin-PE (Invitrogen, San Diego, CA, USA) according to the manufacturer's protocol. At day 10 post challenge, each group of mice was sacrificed and tracheotomy was performed. The lungs were perfused with 5 mL of PBS with 10 U/mL heparin (Sigma) using a syringe with a 25-gauge needle. The lung tissues were homogenized with 3 mL of Iscove's Modified Dulbecco's Medium (IMDM) through 70-µm cell strainers to obtain single-cell suspensions. Centrifugation was conducted afterward, lymphocytes were resuspended in fresh IMDM and washed with fluorescence-activated cell sorting (FACS) buffer (0.5% FBS and 0.09% NaN_3 in PBS). After blocking with anti-mouse CD16/CD32 (BD Biosciences, Franklin Lakes, NJ, USA), cells were stained with anti-CD8a APC (clone 53-6.7, Biolegend, San Diego, CA, USA), anti-CD44 FITC (clone IM7, Biolegend), and D^d/NP(I) tetramer-PE or D^d/NP(V) tetramer-PE. After staining, the cells were washed and fixed with FACS lysing solution for 15 minutes at RT in the dark. Cells were washed with FACS buffer twice and stored at 4℃ in the dark until FACS analysis.