Anti trail

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 following fluorescently conjugated antibodies were used for phenotypic analysis of NK cells: anti-LFA-1 (363404); anti-CXCR3 (353720); anti-PD-1 (329906); anti-CD107a (328612); anti-NKP44 (325116); anti-CD158e1 (312706); anti-CD158d (347006); anti-CD27 (356412); anti-CCR4 (359412); anti-DNAM-1 (338316); anti-CD16 (302040); anti-Granzyme B (515406); anti-CD62L (304806); anti-CD69 (310910); anti-NKP80 (346706); anti-NKP30 (325210); anti-CD158f (341304); anti-CD158b (312612); anti-Tim-3 (345012); anti-CD94 (305504); anti-TIGIT (372706); anti-TRAIL (308206); anti-CD57 (322306); anti-CX3CR1 (341610); anti-NKG2D (320808); anti-Perforin (353310); anti-Ki67 (350504); anti-IFN-γ (506518); anti-CD94 (305506); anti-NKp46 (331916); anti-CTLA4 (369614); anti-CD96 (338416); anti-41BB (309818); anti-CD25 (356108) were purchased from Biolegend. anti-CD159a/NKG2A (FAB1059P) and anti-NKG2C (FAB138G) were purchased from R&D.
The following fluorescently conjugated antibodies were used to identify immune cell types in eNK or PBMC: anti-human Lineage Cocktail (348803); anti-CD56 (362550); anti-CD3 (300430); anti-CD33 (366620); anti-HLA-DR (307606); anti-CD14 (301836); anti-CD19 (302242); anti-CD11b (301322); anti-CD25 (356108); and anti-FOXP3 (320108) were purchased from Biolegend, San Diego, CA, USA.