Chromium-51 Cytotoxicity Assay for NY-ESO-1-Specific T Cells

The SW982 target cells were pretreated with panobinostat, vorinostat or DMSO (solvent control) for 48 h. Specific lysis of SW982 cells by NY-ESO-1-specific T cells was evaluated using a 12 h Chromium-51 (⁵¹Cr) release assay as described previously [19 (link),20 (link)]. In brief, pretreated target cells were labeled with ⁵¹Cr (Hartmann Analytic, Braunschweig, Germany) for 2 h and co-incubated with T cells (effector cells) for 12 h in the absence of HDACi in 96-well U-bottom microplates (Greiner Bio-One) at 37 °C and 5% CO2. An effector to target cell (E:T) ratio of 10:1 i was used. Maximal release was determined by incubating the target cells with 1% Triton X-100 (Merck KGaA, Darmstadt, Germany). Spontaneous release was assessed with medium only. A total of 75 μL of culture supernatant was added to Ultima Gold liquid scintillation cocktail (PerkinElmer, Waltham, MA, USA). Data acquisition was performed on a Wallac Winspectral 1414 liquid scintillation counter (PerkinElmer). All experiments were performed in triplicates. Following formula was used to calculate specific lysis: % specific lysis = (⁵¹Cr release in the test well—spontaneous ⁵¹Cr release)/(maximum ⁵¹Cr release—spontaneous ⁵¹Cr release) × 100.