Celltiter glo reagent

Anchorage-independent growth was measured in 96-well plates. The 6% agarose solution stock was diluted in SUM149 culture medium and 100 μl was used to make the 0.6% bottom layer in opaque with clear bottom 96-well assay plates. After the bottom layer was gelified, a serial twofold dilution of cells was mixed with complete culture media containing agarose at a final concentration of 0.3%. Fifty microlitres per well (corresponding to 250 and 500 cells) were added to the bottom layer. Cells were incubated for 21 days at 37 °C in 5% CO₂ atmosphere. Unless otherwise stated, cell number was quantified using CellTiter-Glo reagent (Promega) as follows: 75 μl of CellTiter-Glo reagent (Promega) and luminescence was recorded after 10 min of incubation time using a microplate luminometer.

Intracellular ATP was measured using CellTiter-Glo Reagent (Promega, Madison, WI, USA). Briefly, cells were harvested, quantified, and then lysed with lysis buffer. Fifty microliters from each diluted sample was mixed with an equal volume of CellTiter-Glo Reagent, and the sample was incubated at room temperature for 10 min and analyzed using a GloMax luminometer (GloMax 96 Microplate Luminometer; Promega).
After oridonin treatment, the medium was removed from the cells, and lactate levels in the extracellular medium were measured using a l-lactate Colorimetric Assay Kit (Abcam, Cambridge, UK). Then, the intracellular lactate levels were measured in the cell lysates. Data were normalized to the final cell counts.

High-throughput combinatorial screens were performed as previously described (45 (link)) with some modifications. The initial screen in H446 cells was performed in a 6 × 6 matrix layout by combining JQ1 with a MIPE 4.0 library, which consists of 1,912 FDA-approved oncological therapeutics and investigational agents. The viability endpoint of the initial screen was measured using the CellTiterGlo reagent (Promega) 72 hours after drug treatment. The synergy of various drug combinations was assessed using the excess Highest Single Agent (HSA) method, the response heat map, and the Δ Bliss heat map (46 (link), 47 (link)).
The second screen was performed in 4 SCLC cell lines of 2 molecular subtypes (SCLC-N: H446, COR-L279, and SCLC-21H; and SCLC-A: H187) in a 10 × 10 matrix by combining JQ1 or iBET-762 with 1 of the forty drugs selected from the first screen. The endpoints of the second screen were caspase 3/7 activity measured using the CaspaseGlo 3/7 assay (Promega) at 8-hour and 16-hour intervals, as well as cell viability measured using the CellTiterGlo reagent at the 72-hour interval.

Cells were treated with AITC and capsaicin dissolved in DMSO to different concentrations (5 µM, 10 µM, 20 µM, 50 µM, 100 µM, and 200 µM). The DMSO concentrations of the solvent-treated control cells were adjusted to similar concentrations as the concentrations of the treatments (2%, 1%, 0.5%, 0.2%, 0.1% and 0.05%). For the pre-treatment, cells were treated separately with 10 µM HC-030031 and capsazepine for 10 min prior the addition of the agonists AITC and capsaicin, respectively. K7M2 cell viability was assessed using a CellTiter-Glo^® Luminescent Cell Viability Assay (CTG, Promega, Madison, WI, USA) mixture as recommended by the manufacturer. Cells were seed in 96-well tissue culture plates at a density of 5000 cells/well in 100 µL of media and cultured for 24 h. On the following day, compounds were added at the above-mentioned concentrations and cells were incubated for 48 h. When this incubation was completed, the plate and its contents were balanced at room temperature for approximately 30 min. Promega CellTiter-Glo^® Reagent (Promega, Madison, WI, USA) was added in a volume equal to the cell culture medium present in each well. An ATP-based luminometric measurement of the metabolically active cells in the culture were determined by an EnSpire^® Multimode Plate Reader (Waltham, MA, USA) as relative light units (RLUs). Viability was calculated as follows:

For ATP measurement, the CellTiter-Glo Assay Kit (Cat # G7570; MilliporeSigma) was used according to the manufacturer’s protocol. First 100 µL of sorted CD4⁺ T cells (3 × 10⁵) were stimulated with anti-CD3 (2 µg/mL) and anti-CD28 antibodies (2 µg/mL) for 24 h at 37 °C in a 96-well opaque-walled flat bottom plate. After stimulation, 100 µL of Promega CellTiter-Glo reagent (Promega Corporation, WI, USA) was added to each sample well, incubated at 20–22 °C for 10 min, and mixed by pipetting. The luminescence was measured using a Tecan Infinite F200 fluorescence microplate reader (Tecan Group Ltd., Männedorf, Switzerland). The percentage of ATP change in Prkaa1^T-KO or Sirt1^T-KO CD4⁺ T cells was calculated relative to the luminescence value of WT CD4⁺ T cells.