Fitc anti vγ2 7a5

Cultured cells were washed with PBS, stained with Zombie Fixable Viability Kit (Biolegend), followed by staining with monoclonal Abs against special surface markers. For cell memory state analysis, cells were incubated with PB-anti-CD3 (SP34-2, BD), FITC-anti-Vγ2 (7A5, Thermo Scientific), PE-anti-Vδ2 (B6, Biolegend), BV785-anti-CD45RA (HI100, Biolegend), PE/Cy7-anti-CD27 (O323, Biolegend), PE/Cy5-anti-CD28 (CD28.2, Biolegend) for 20 min at room temperature in dark. For phenotyping of special surface markers, cells were incubated with PB-anti-CD3 (SP34-2, BD), FITC-anti-Vγ2 (7A5, Thermo Scientific), PE-anti-Vδ2 (B6, Biolegend), APC-anti-CCR5 (J418F1, Biolegend), PE/Cy5.5-anti-LFA-1 (TS1/18, Biolegend) for 20 min at room temperature in dark. Then cells were washed and fixed by fixing buffer (2% formalin in PBS), analyzed on an LSR Fortessa flow cytometer (BD).

PBMCs cultured for 7 days were firstly treated with/without 40 ng/ml HMBPP, pulsing PE/CF594-anti-CD107a (H4A3, Biolegend), and BFA (GolgiPlug, BD) for 6 h. Then cells were stained with Zombie Fixable Viability Kit (Biolegend), incubated with PB-anti-CD3 (SP34-2, BD), FITC-anti-Vγ2 (7A5, Thermo Scientific), PE-anti-Vδ2 (B6, Biolegend) for 20 min at room temperature in dark. Cells were permeabilized for 30 min at 4 degrees (Cytofix/Cytoperm, BD). After wash, cells were incubated with BV711-anti-IFN-γ (4S.B3; Biolegend), PE/Cy7-anti-TNF-α (Mab11, Biolegend), Percp/Cy5.5-anti-GM-CSF (BVD2-21C11, Biolegend) for 30 min at room temperature in dark. Then cells were washed and analyzed on an LSR Fortessa flow cytometer (BD). CD107a was used to assess degranulation of cytotoxic molecules; IFN-γ, TNF-α, and GM-CSF was used to evaluating anti-mycobacteria effector function. Flow data were analyzed by FlowJo (TreeStar).