Fitc anti mouse cd8 clone kt15

Isolated lung cells were stimulated with a peptide (A-NP_147–155 or B-NP_166–174) at 37°C for 5 h in the presence of brefeldin A (Thermo Fisher Scientific). They were blocked using a purified rat anti-mouse CD16/CD32 Fc blocker (BD Pharmingen, San Diego, CA, USA). Thereafter, the cell surface was stained with APC/Cy7 anti-mouse CD3 (Clone 17A2; BioLegend) and FITC anti-mouse CD8 (clone KT15; MBL Life Science, Woburn, MA, USA). After 30 min, they were intracellularly stained for cytokine production using a BD Cytofix/Cytoperm kit (BD Biosciences, Heidelberg, Germany) in accordance with the manufacturer’s instructions. For IFN-γ and TNF-α measurements, APC anti-mouse IFN-γ (clone XMG1.2; BioLegend) and PE anti-mouse TNF-α (clone MP6-XT22; BioLegend) were added to the samples.

Isolated cells were blocked using a purified rat anti-mouse CD16/CD32 Fc blocker (BD Pharmingen) and 5 μg/ml streptavidin (Sigma) at 4°C for 20 min. Subsequently, the cells were stained with APC/Cy7 anti-mouse CD3 (clone 17A2; BioLegend), FITC anti-mouse CD8 (clone KT15; MBL Life Science), eF450 anti-mouse CD103 (clone 2E7; Thermo Fisher Scientific), PE/Cy7 anti-mouse CD69 (clone H1.2F3; BioLegend), APC anti-mouse CD62L (MEL-14; BioLegend), PE A-NP-specific K^d/NP_147–155 tetramer, and B-NP-specific D^d/NP_166–174 tetramer at 4°C for 30 min. Continually stained cells were fixed in a FACS-lysing solution (BD Pharmingen) at 25°C for 20 min and analyzed using Beckman CytoFLEX S (Beckman Coulter, San Jose, CA, USA) and FlowJo Software (TreeStar Inc., Ashland, OR, USA).