Anti puromycin af488

An amount of 0.2 × 10⁶ NK cells/well were plated in 100 µL IMDM media and incubated in the presence or absence of Oligomycin (1 µM), 2-DG (100 nM) or Oligomycin + 2-DG for 30 min at 37 °C and 5% CO₂. Next, 100 µL IMDM media containing Puromycin (10 µg/mL) was added to each well and incubated for 20 min at 37 °C and 5% CO₂. Then, cells were washed with ice-cold PBS, and Fc-Block for 5 min at 4 °C was performed followed by a surface staining using zombie NIR (1:700; BioLegend), anti-CD56 BV421 (1:100; Clone:NCAM16.2; BD Biosciences) and anti-CD16 PE-Dazzle (1:100; Clone:3G8; BioLegend) for 30 min at 4 °C. Cells were subsequently washed, fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (Invitrogen^TM, Waltham, MA, USA) and then intracellular staining using anti-Puromycin AF488 (1:200; Clone:12D10; Sigma-Aldrich, St. Louis, MO, USA) and anti-pmTOR PE-Cy7 (1:100; Clone:MRRBY; Thermo Fisher Scientific) was performed for 30 min. Lastly, cells were washed again and then measured on a Cytek Aurora flow cytometer. Gylcolytic dependency, mitochondrial dependency, glycolytic capacity and FAO (fatty acid oxidation) and AAO (amino acid oxidation) capacity were calculated using the formula as already described by Argüello et al. [15 (link)]

Assays were performed as described in [16 (link)]. In brief, NK cells were incubated in the presence or absence of Oligomycin (1 µM), 2-DG (100 nM) or Oligomycin + 2-DG for 30 min. Next, Puromycin (10 µg/mL) was added for 20 min followed by a wash using ice cold PBS. After an Fc-Block for 5 min, surface staining was performed using Zombie NIR (BioLegend), anti-CD56 BV421 (Clone:NCAM16.2; BD Biosciences), and anti-CD16 PE-Dazzle (Clone:3G8; BioLegend). Cells were then washed, fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) followed by intracellular staining using anti-Puromycin AF488 (Clone:12D10; Sigma-Aldrich) and anti-pmTOR PE-Cy7 (Clone:MRRBY; Thermo Fisher Scientific) and analysis using a Cytek Aurora flow cytometer. Glycolytic dependency, mitochondrial dependency, glycolytic capacity, and FAO (fatty acid oxidation) and AAO (amino acid oxidation) capacity were calculated using the formula as already described by Argüello et al. [18 (link)].