Anti tcrγδ

Ninety days after the initial FTG, mice were anesthetized with Avertin (250 mg/kg, i.p.) and blood was collected by cardiac puncture using a heparinized syringe. Each mouse was perfused transcardially using ice cold PBS solution and the brain was removed. The right hemisphere was homogenized and incubated in RPMI-1640 (Corning) containing 1 mg/ml of collagenase/dispase (Roche) and 10 mg/ml of DNase I (Roche) for 45 min at 37°C. The cell suspension was filtrated through a 70-μm cell strainer, washed and applied to a Percoll (GE Healthcare) gradients of 30% and 70% using the centrifugation at 500 g for 20 min. The interphase between the gradients was collected. Cells were stained with anti-CD45 (clone: 30-F11, BioLegend 103139), anti-CD4 (clone: RM4-5, BioLegend 100516), anti-CD8α (clone: 53–6.7, BioLegend 100737) and anti-TCRγδ (clone: GL3, BioLegend 118124). An amine reactive Live/Dead Aqua viability stain (Invitrogen L34966) was used to identify only live cells. Fluorescence-minus-one controls were used to distinguish positively stained cells for each antibody. The cells were analyzed using CytoFLEX (BECKMAN COULTER). The data were analyzed using FlowJo software (Tree Star, Inc.).

CytoTox 96^® Non-Radioactive Cytotoxicity Assay kits (Promega), which are based on the colorimetric detection of the released enzyme lactate dehydrogenase (LDH), was used to determine specific cytotoxicity. Vγ9Vδ2 T cells (Effector, E) were co-cultured with LX-2 cells (Target, T), which were pretreated with N-BPs for 4 h at specific E/T ratios for 4 h (indicated in figure captions). In some cases, after 4 h co-cultured, the number and area of Vγ9Vδ2 T cells clusters were determined using IncuCyte (Essen BioScience) image analysis software. Three different locations per well were imaged with a 10 × objective lens. Clusters were defined as cell aggregates occupying an area at least 300 µm² and were displayed as the number and area of clusters per well. In some experiments, Transwell^® units (pore size 0.4 µm, Corning) were used to separate Vγ9Vδ2 T cells from LX-2 cells. In some cases, the neutralization antibodies anti-NKG2D (10 µg/mL, BD), anti-FasL (10 µg/mL, Biolegend), anti-TRAIL (10 µg/mL, Biolegend), anti-TCR γδ (10 µg/mL, Biolegend), and their relevant isotype controls, were individually added in the co-cultures to block the NKG2D-, FasL-, TRAIL-, and TCR γδ- mediated pathways. To block perforin and granzyme B pathways, the perforin inhibitor Concanamycin A (CMA, Selleck) (1 µg/mL) and granzyme B inactivator BCL-2 (1 µg/mL, R&D Systems) were used (15 (link)).

The samples were treated with trypsin, permeabilized by 0.5% PBST, peroxided by hydrogen peroxide solution, and blocked in goat serum. The following antibodies were used: anti-TCR γδ (BioLegend catalog no. 118101; BioLegend catalog no. 331201), anti-CD206 (Proteintech catalog no. 18704-1-AP), anti-IL-17 (Proteintech catalog no. 26163-1-AP), anti-PD-1H (BioLegend catalog no. 143702), and anti-pSTAT3 (Cell Signaling Technology catalog no. 9145). The secondary antibodies (BioLegend catalog no. 405510; Abcam catalog no. ab6717; Abcam catalog no. ab150115) and 4′,6-diamidino-2-phenylindole (DAPI) (Solarbio catalog no. C0065) were incubated successively. After staining, slides were examined on an Olympus confocal microscope (FV31S-SW V2.4 software).