Tandem signal enhancer

For the analysis of activating receptor expression by cytotoxic NK cells (CD3-; CD56dim), 5 × 10⁶ human PBMCs were treated with the respective chemotherapeutic compound and incubated for 72 h (37 °C/5% CO₂). Then cells were harvested and 1 × 10⁶ live cells were washed with wash buffer, followed by incubation with 10 µL of Tandem Signal Enhancer (Miltenyi Biotec). Incubation with the following antibodies in a total volume of 100 µL was conducted for 20 min at RT: CD3-VioGreen (REA613, 1:200), CD56-APC-Vio770 (REA196, 1:200), CD226-VioBlue (REA1040, 1:50); CD335 (NKp46)-Vio Bright B515 (REA808, 1:50), CD337 (NKp30)-PE (REA823, 1:75), CD336 (NKp44)-APC (REA1163, 1:75), and CD314 (NKG2D)-PE-Vio 770 (REA1228, 1:75), all from Miltenyi Biotec. As isotype controls served respective REA controls (Miltenyi Biotec, REA293). To exclude dead cells from analysis, 0.25 µg propidium iodide solution was added prior to acquisition. At least 20,000 CD3−/CD56+ cells were analyzed for each sample.

Expression of cell surface markers on differentiated T cells was determined using the MACSQuant^® flow cytometer. Briefly, cells were harvested from culture at indicated time points and incubated with the appropriate concentration of monoclonal antibody (Table S1) with Tandem Signal Enhancer (Miltenyi Biotec. Inc., Bergisch Gladbach, Germany) in flow cytometry staining buffer (dPBS, 0.5% bovine serum albumin, 0.5 mM EDTA) for 10 min at 4 °C. Cells were washed once by centrifugation, and propidium iodide (Miltenyi Biotec. Inc., Bergisch Gladbach, Germany) was added to exclude dead cells. Data were analyzed using the FlowLogic^™ software (Miltenyi Biotec. Inc., Bergisch Gladbach, Germany). Staining controls included: unstained cells, peripheral blood mononuclear cells and isotype-matched control antibodies. All antibodies, including isotype controls, were purchased from Miltenyi Biotec. Inc. (Supplementary Table S1).