SCENITH™

Cells were plated at 20 × 10⁶ cells/ml in 96-well plates. After activation of γδ T cells, cells were treated for 30 minutes at 37°C, 5% CO₂ with Control (Co), 2-Deoxy-D-Glucose (DG; 100mM; Sigma-Aldrich), Oligomycin (O; 1μM; Sigma-Aldrich) or a combination of both drugs (DGO). Puromycin (Puro, 10μg/ml; Sigma-Aldrich) is added for 15min at 37°C. SCENITH™ kit (http://www.scenith.com) containing all reagents and protocols were developed by Dr. Rafael Argüello, (CIML). Cells were washed in cold PBS and stained with primary conjugated antibodies against different surface markers (as described above) for 15min at 4°C in FACS buffer (PBS 1X 5% FCS, 2mM EDTA). After washing with FACS buffer, cells were fixed and permeabilized using Cytofix/Cytoperm™ (BD) following manufacturer’s instructions. Intracellular staining of puromycin using the anti-Puro monoclonal antibody (1:600, Clone R4743L-E8) was performed by incubating cells during 30min at 4°C diluted in Permwash. Experimental duplicates were performed in all conditions.