Fitc conjugated anti mouse tcrβ clone h57 597

The ear draining lymph nodes were digested with 3 mg/mL collagenase type IV (Invitrogen) and 1 mg/mL DNAse type I (Sigma) for 45′ at 37°C, and filtered through a 70 µm gauge strainer (Becton Dickinson). The cells were counted with a hemacytometer and labelled for FACS analysis. Fc receptors were blocked with the mAb 2.4G2. Cells were stained for cell surface antigens with the following reagents: FITC-conjugated anti-mouse TCRβ (clone H57-597, eBioscience), Brilliant Violet 421-conjugated anti-mouse CD8α (clone 53-6.7, Biolegend), Brilliant Violet 785-conjugated anti-mouse CD4 (clone RM4-5, Biolegend). Cells were analyzed with a Gallios FACS machine (Beckman Coulters) and the data were processed with Kaluza software (Beckman Coulters). The number of cells per population was calculated by multiplying the total number of lymph nodes cells with the percentage of cells of interest. For T cell restimulation, draining lymph nodes cells were incubated at 37°C under 5% CO₂ for 72 hours in the presence of UV-irradiated L. major (ratio 5∶1, cell∶parasite). Supernatant were collected and the levels of IL-4 and IFN-γ were measured by ELISA (eBioscience) or CBA (Becton Dickinson) according to the manufacturer instructions.

Lymphocytes isolated from the lung were cultured in 1640 medium and 10% FBS. Before FACS analysis, cells were stimulated with 25 ng/mL phorbol 12-myristate 13-acetate (PMA, ebioscience), 1ug/mL ionomycin (ebioscience), and Brefeldin A (BFA ebioscience) at 37 °C for 4 hours. Isolated lymphocytes were stained with FITC-conjugated anti-mouse TCRβ (clone H57-597, eBioscience), and PE-cy5-conjugated anti-mouse TCRγδ (clone eBioGL3, eBioscience). After surface staining, cells were washed, fixed, and permeabilized for intracellular staining with PE-conjugated anti-mouse IL-17A (clone eBio17B7, eBioscience). All fluorophore-conjugated antibodies and the corresponding isotypes were from eBioscience. The stained cells were used for flow cytometric analysis (FC500, Beckman).