Ultima gold liquid scintillation cocktail

[1-¹⁴C] acetate labeling was performed according to the method of Koo et al. (2004 (link)). In brief, half leaves were incubated in 25 mM Na-Morpholinoethanesulfonic acid (MES, pH 5.7) buffer containing 0.01% w/v Tween-20 as wetting agent under illumination (180 μmol photos m⁻²s⁻¹) at 25°C. Labeling was initiated by the addition of 10 μCi of sodium [1-¹⁴C] acetate solution (58 mCi/mmol, American Radiolabeled Chemicals). Labeling was terminated by the removal of the medium after which the leaf material was washed 3x with deionized distilled water. Total lipids were extracted and separated as described above. Lipids were suspended in 2 mL of Ultima Gold liquid scintillation cocktail (PerkinElmer) and radioactivity was quantified in CPM using scintillation counter (Packard BioScience).

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.

Glycerol tri [³H]oleate ([³H]triolein [³H]TO; NET431L005MC, PerkinElmer)-labeled TG-rich lipoprotein (TRL)-like particles were prepared as previously described,²⁴ and [¹⁴C]deoxy-d-glucose ([¹⁴C]DG; EC495A250UC, PerkinElmer) was added to the emulsion (5:1 ³H:¹⁴C activity ratio). Directly after the collection of 4-h fasted blood, mice (cohort 1) were intraperitoneally injected with 2 g kg⁻¹d-glucose to induce a standardized fed state while avoiding the production of endogenous GIP and GLP1 by the intestine. Half an hour later, mice were intravenously injected with the mixture of [³H]TO-labeled TRL-like particles (1 mg TG per mouse) and [¹⁴C]DG in 200 μL PBS, and killed by CO₂ inhalation 15 min thereafter. After collecting blood via heart puncture to assess ALT activity (MAK052, Sigma–Aldrich), mice were perfused with ice-cold PBS. Collected tissues (max. 200 mg) were weighed and dissolved overnight at 55 °C in 500 μL Solvable (Perkin Elmer), after which 5 mL Ultima Gold liquid scintillation cocktail (PerkinElmer) was added to determine ³H and ¹⁴C activity using a Tri-Carb 2910 TR Low Activity Liquid Scintillation Analyzer (PerkinElmer). Uptake of [³H]TO- and [¹⁴C]DG-derived radioactivity by organs was expressed as the percentage of injected dose per gram of wet tissue.

HepG2 cells were incubated with sodium citrate (1 or 5 mM) and CoCl₂ (100 μM, Sigma-Aldrich, St. Louis, MO, USA). After incubating for 24 h, cells were treated for 1 h with 0.5 μCi ¹⁴C-DG (Perkin Elmer, Waltham, MA, USA) in DMEM medium (Gibco, cat # 11966-025, Waltham, MA, USA) with 5 mM glucose, or 0.1 μCi ¹⁴C-citrate (PerkinElmer) in a reaction buffer containing 25 mM HEPES (pH 7.5), 140 mM sodium chloride, 5.4 mM potassium chloride, 0.8 mM magnesium sulfate, 1.8 mM calcium chloride, 25 mM Tris-HCl, and 5 mM D-glucose for 2 h. After washing with PBS, the cells were lysed using 0.1 N NaOH lysis buffer (Sigma-Aldrich) for 2 h. The cell lysates were collected in 6 mL pony vials (Perkin Elmer) containing 5 mL Ultima Gold liquid scintillation cocktail (Perkin Elmer) and measured using a liquid scintillation counter (Perkin Elmer). The measured count per minute was normalized to the protein concentration determined using a bicinchoninic acid protein assay kit (Thermo Fisher, Waltham, MA, USA).

Radioactively labelled (tritium) thymidine was used to measure the proliferation of MDA-MB-231 cells by the incorporation of [³H] thymidine into the DNA of dividing cells. Cells were seeded at a seeding density of 2 × 10⁴ cells in 24 well plates. Following treatment, cells were labelled with 0.1 μCi [³H] thymidine per well, incubated with 5% trichloroacetic acid at 4°C for 10 minutes, followed by a 1 hour incubation with 1 M sodium hydroxide. The resulting suspension was added to a vial containing 2 mL ultima gold liquid scintillation cocktail (Perkin Elmer Beaconsfield, Bucks, UK) and incorporated counts were measured using a Beckman Scintillation Counter LS6500. Data were recorded as disintegrations per minute (DPM).

The specific anti-tumor efficacy of NY-ESO-1-specific T cells was measured using a 12-h Chromium-51 (⁵¹Cr; Hartmann Analytic, Braunschweig, Germany) release assay. SW982 cells (NY-ESO-1+ HLA-A2+) served as target cells, whereas SYO-1 cells (NY-ESO-1- HLA-A2-) were used as negative control. Cell lines were labeled with ⁵¹Cr for 2 h and co-incubated with NY-ESO-1 specific T cells or non-transduced T cells (effector cells) for 12 h using effector to target cell (E:T) ratios of 10:1, 5:1, 2.5:1 and 1:1 (5:1 only for non-transduced T cells) in 96-well U-bottom microplates (Greiner Bio-One) at 37 °C and 5% CO2. Maximum release and spontaneous release were determined by incubating the target cells with 1% Triton X-100 (Merck KGaA, Darmstadt, Germany) and medium alone, respectively. In addition, 75 μL of culture supernatant was diluted in Ultima Gold liquid scintillation cocktail (PerkinElmer, Waltham, MA, USA) and measured on a γ-counter (PerkinElmer). All experiments were performed in triplicates. Specific lysis was calculated according to the following formula: % specific lysis = (⁵¹Cr release in the test well—Spontaneous ⁵¹Cr release)/(maximum ⁵¹Cr release—Spontaneous ⁵¹Cr release) × 100.

The ⁸⁶Rb⁺ uptake assay procedure was based on previously established protocols [41 (link)]. More specifically, three hr before each experiment, the cell culture medium was replaced with DMEM containing 0.2% FBS. Following serum deprivation, the cells were treated with ouabain (2 mM) (O3125; Sigma-Aldrich, Oakville, ON, Canada) and/or SST14 (50 μM) or with vehicle (control). After incubation for 15 min at room temperature, 2 μCi ⁸⁶RbCl in water (NEZ072; PerkinElmer, Woodbridge, ON, Canada) was added to each well and the cells were incubated for another 10 min at 37°C. The supernatants were then removed, and cells were washed four times with 1 mL ice-cold wash buffer (100 mM MgCl₂, 10 mM HEPES, pH 7.4) before lysis in 500 μL buffer containing 1% NP40, 150 mM Tris (pH 8.3), and 150 mM NaCl. 250 μL aliquots of the cell lysates were transferred to vials containing 10 mL Ultima Gold liquid scintillation cocktail (6013326; PerkinElmer, Woodbridge, ON, Canada) and assayed using a liquid scintillation counter (LS6500; Beckman Coulter; Mississauga, ON, Canada). Additional 10 μL aliquots were used for protein concentration determination using BCA reagents (23228 and 1859078; Thermo Fisher Scientific, Burlington, ON, Canada).