Celltitre glo luminescent cell viability assay

Cells were stripped of E2 by culture in RMPI1640 containing 10% DCC for 48 h prior to seeding into 96-well tissue culture plates. Monolayers were allowed to acclimatise for 24 h prior to treatment with RPMI1640 + 10% DCC containing increasing concentrations of drugs. The medium was replaced after 3 days, and cells were cultured for a total of 5–6 days. Cell viability was determined using the CellTitre-Glo® Luminescent Cell Viability Assay (Promega), according to the manufacturer’s protocol. Values were expressed as % viability relative to the vehicle-treated control. Spheroid cultures were generated by seeding 2500 cells per well of a 96-well ultra-low attachment plate (Costar). Plates were spun at 900×g for 10 min. Spheres were formed over 72 h and subsequently treated with the drugs as indicated for 7 days. Proliferation was assessed using Celigo S (Nexcelom Bioscience).

The assay was performed according to the manufacturer kit (Cell Titre Glo Luminescent Cell Viability Assay, Promega). CLL cells were plated 3 × 10⁶/mL in their culture medium within a 96‐opaque‐walled plate in a volume of 100 μL. Control wells were prepared adding only medium. Then, an equal amount of Cell Titre Glo Reagent was added to each well and the two parts were mixed for 2 min on a shaker. The multiwell was then left 10 min at room temperature to stabilize the signal. Finally, luminescence was recorded with a Luminometer. The results were compared to an ATP standard curve previously generated.

BAY-320 plus Paclitaxel combination studies were conducted with HeLa and NCI-H1299 cells. Cells were plated into 384-well plates at 600 (HeLa) or 700 (NCI-H1299) cells per well. After 24 hr, cells were treated with BAY-320 (concentration range, 1E-07 M to 1E-05 M) and Paclitaxel (concentration range, 1E-09 M to 1E-07 M) for single compound treatments, and in nine different fixed-ratio combinations of BAY-320 (D1) and Paclitaxel (D2) (0.9xD1+0.1xD2, 0.8xD1+0.2xD2, 0.7xD1+0.3xD2, 0.6xD1+0.4xD2, 0.5xD1+0.5xD2, 0.4xD1+0.6xD2, 0.3xD1+0.7xD2, 0.2xD1+0.8xD2, 0.1xD1+0.9xD2). Cell viability was assessed after 96 hr exposure, using the Cell Titre-Glo Luminescent Cell Viability Assay (Promega). IC₅₀ values were determined by means of a 4-parameter fit after normalization of measurement data to vehicle (DMSO)-treated cells (=100%) and readings taken immediately before compound addition (=0%). IC₅₀ isobolograms were plotted with the actual concentrations of the two compounds on the x- and y-axis, and the combination index (CI) was calculated according to the median-effect model of Chou-Talalay (Chou, 2006 (link)). A CI of ≤0.8 was defined as more than additive (i.e. synergistic) interaction, and a CI of ≥1.2 was defined as antagonistic interaction.

MCF7 cells were reverse-transfected. siRNA upon receipt were prepared as 20 mM stocks and stored at −20 °C (ON-TARGETplus Human FOXA1 siRNA [Entrez 3169]; ON-TARGETplus Non-targeting Control siRNAs from Horizon). For 24-well plates, the siRNA was diluted to a concentration of 2 mM using serum-free RPMI1640 and 6 ml was added to 994 ml of serum-free RPMI1640 to give 12 pmol per well of a 24-well plate. Lipofectamine RNAiMAX (Thermofisher) 1 ml was added to each well. Plates were gently mixed and incubated for 10–20 min at room temperature. Cells were diluted in RPMI1640 to a final concentration of 60,000 cells/ml. About 500 ml of cells was added to each well, giving a final number of 30,000 per well of a 24-well plate. The following day the medium was changed to the relevant selection and incubated for 72 h. Monolayers were then washed and subjected to immunoblotting. For 96-well plates, each well was treated with 2.4 pmol per well for each siRNA and 0.2 ml of Lipofectamine RNAiMAX in 20 ml of RPMI1640. Cells were diluted to 15,000/ml and 100 ml added to each well to give a final concentration of 1500/well. Plates were incubated overnight and then the medium was changed to the appropriate selection. Plates were incubated for a further 5 days and proliferation was assessed using CellTitre-Glo® Luminescent Cell Viability Assay (Promega).

Meningioma cells were plated in technical triplicates and biologic duplicates separated by at least one passage of each cell line in Corning 96-well white-walled plates. Cells were treated with palbociclib (InvivoChem catalogue No. V1531; 10, 25, 50, 100, 1000, 10,000 nM), or abemaciclib (InvivoChem catalogue No. V1547; 10, 25, 50, 100, 1000, 10,000 nM) for 10 consecutive days. A medium-only control was used for each replicate of each drug treatment. Two days after completion of treatment, CellTitre-Glo luminescent cell viability assay was used in accordance with the manufacturer’s instructions (Promega, catalog no. G7570). Cells were incubated for 10 min with the CellTitre-Glo reagent and luminescence was measured using a 96-well plate reader (GloMax-96 microplate luminometer; Promega). Background luminescence was measured in blank wells with medium without cells. For analysis, concentrations were log-transformed and all luminescence values were normalized based on the highest viability of each technical replicate as the maximum (100% viability), and the medium-only blank wells as the minimum (0% viability).

Epigallocatechin-gallate (EGCG, Sigma-Aldrich, St. Louis, MO, USA, E4143) and honokiol (HNK, Sigma-Aldrich, St. Louis, MO, USA, H4914) were dissolved in DMSO at 100 mM and 10 mM, respectively, and stored at −20 °C. To determine cell viability, EGCG and HNK were serially diluted onto 1 × 10⁴ A549 cells for 26 h. CellTitre-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA) reagent was then added to cells following manufacturer’s instructions. Values were normalised to DMSO controls. For infection assays, A549 cells were treated with EGCG or HNK 2 h prior to and/or throughout a 24 h infection with ZIKV-Nanoluc (multiplicity of infection (MOI) 0.1). Where specified, ZIKV-Nanoluc was incubated with either EGCG or DMSO for 2 h in DMEM plus 2% FBS prior to infection of cells. To measure activity of Nanoluc expressed by a virus, cells were first lysed in Passive Lysis Buffer (Promega, Madison, WI, USA) before detection with Nano-Glo Luciferase Assay System plate reader (Promega, Madison, WI, USA). Values were normalised to infected cells treated with DMSO only. All drug treatments were performed in DMEM supplemented with 2% FBS at 37 °C with 5% CO₂.

Cells lines were seeded in 96-well plates. Monolayers were allowed to acclimatise for 24-hours prior to treatment with drug concentrations and combinations, as indicated, for 6-days with a medium change on day 3. Cell viability was determined using CellTitre-Glo^® Luminescent Cell Viability Assay (Promega), according to the manufacture’s instructions.