Anti mouse cd3 fitc clone 17a2

Lung and lymph nodes were examined for intracellular cytokine production. Single cells were obtained as described above. Samples were then stimulated with 10 μg/ml H37Rv cell lysate (BEI NR-14822), 5 μg/ml ESAT6 peptide array (BEI NR-34824), and 5 μg/ml CFP10 peptide array (BEI NR-34825) in RPMI media for 3 h in a 37 °C, 5% CO₂ humidified incubator. Stimulated cells were then washed, and surface stained with anti-mouse CD3-FITC (clone 17A2, BD Pharmingen), anti-mouse CD4-V450 (clone RM4-5, BD Horizon), and anti-mouse CD8-PE (clone RM4-5, BD Pharmingen). Subsequently, cells were fixed and permeabilized using BD Cytofix/Cytoperm solution kit (BD 554714) as per the manufacturer’s instructions. Permeabilized cells were stained for intracellular cytokines using anti-mouse IL-17A-AF647 (clone TC11-18H10, BD Pharmingen) and IFNγ-PECy7 (clone XMG1.2, BD Pharmingen). All antibodies were used at a 1:50 dilution. Samples were acquired using the LSRFortessa X-20 and analyzed using FlowJo software (v10.6.1).

Central and effector memory T cells were assessed twenty-eight days post-challenge, as previously described [33 (link)]. Total splenocytes were plated at 5 × 10⁵ cells in a 96-well round-bottom (Costar) and stimulated with SLA (50 μg/mL) at 37 °C with 5% CO₂. Past 5 days of antigen-specific stimulation, samples were blocked with anti-mouse CD16/CD32 (BD Pharmingen^TM) (0.5 μg/well) and stained with markers related to memory T cells phenotype, anti-mouse CD3 FITC (clone 17A2, BD Pharmingen^TM), anti-mouse CD4 BV605 (clone RM4-5, BD Horizon^TM), anti-mouse CD8α PerCPCy5.5 (clone 53-6.7, BD Pharmingen^TM), anti-mouse CD44 APC (clone IM7, BD Pharmingen^TM), anti-mouse CD45RA BV711 (clone 14.8, BD Pharmingen^TM), anti-mouse CD62L AF700 (clone MEL-14, BD Pharmingen^TM), anti-mouse CD127 BV510 (clone SB/199, BD Horizon^TM), and anti-mouse CD197 BV421 (clone 4B12, BD Horizon^TM). The results are expressed by the frequency of the SLiAg-stimulated culture.