Pe cy7 cd69 or pe cd69

Freshly isolated PBMC from CCLm group were stimulated with PMA (50 ng/ml)/ionomicyn (10⁻⁶M) for 6 h (positive control) or SLA (10 μg/ml) or rLmlRAB or rLmlRABC (10 μg/ml) for 120 h, or kept with medium alone. Cells were treated with Golgistop (BD Biosciences) for the last 6 h of culture, then washed and incubated with antibodies: FITC CD3, PerCPcy5.5 CD4, APC-H7 CD8 or PerCPcy5.5 CD8, and PE-Cy7 CD69 or PE CD69 (BD Biosciences), for 20 min at 4 °C. For intracellular IFN-γ detection, cells were fixed and permeabilized using BD Cytoperm/cytofix plus kit (BD Biosciences) according to manufacturer’s instructions and labelled with PE-anti-IFN-γ mAb (intracellular formulation) (BD Biosciences). BD™ CompBeads Set Anti-Mouse Ig, κ (Anti-Mouse Ig, κ/Negative Control (FBS) Compensation Particles Set) (BD Biosciences) was used for compensation controls. Analysis was performed with DIVA software.

Freshly isolated PBMC were stimulated with PMA (50 ng/ml)/ionomicyn (10⁻⁶ M) or PHA at 10 µg/ml for 6 h (positive control) or with TSLA at 10 µg/ml or with LaPSA-38S at 10 µg/ml for 120 h and, or kept with medium alone. Cells were treated with Golgistop (BD Biosciences) for the last 6 hours of culture, then washed and incubated with antibodies: FITC CD3, PerCPcy5.5 CD4, APC-H7 CD8 or PerCPcy5.5 CD8, and PE-Cy7 CD69 or PE CD69 (BD Biosciences), for 20 minutes at 4°C. For intracellular IFN-γ detection, cells were fixed and permeabilized using BD Cytoperm/cytofix plus kit (BD Biosciences) according to manufacturer's instructions and labeled with PE-anti-IFN-γ mAb (intracellular formulation) (BD Biosciences). Analysis was performed with FACSCalibur using CellQuest Pro software or FACS canto II flow cytometer using DIVA software. FITC mouse IgG1, PerCPCy5.5 mouse IgG1, mouse IgG1PE, PE mouse IgG2a (intracellular formulation) (Biosciences) were used as isotype controls for acquisition by FACSCalibur. For acquisition by FACSCanto, BD CompBeads Set Anti-Mouse Ig, κ (Anti-Mouse Ig, κ/Negative Control (FBS) Compensation Particles Set) (BD Biosciences) were used.