Synergy mx multi format microplate reader

For MTT assays, 3 × 10³ ovarian cancer cells (COV-413B, ADR-RES, OVCAR-3, IGROV-1, or SK-OV-3) were plated in triplicate in a volume of 100 µL on 96-well plates. After 48 h, EPZ-5676, EPZ004777, and SGC0946 were added as indicated. Cell viability was evaluated after 3 days of treatment. To measure cell viability, 20 µL 5 mg/mL MTT solution dissolved in 1× PBS was added to each well of the 96-well plate and incubated for 1 h at 37 °C. The MTT solution was then gently removed, and 100 µL DMSO was added to each well. After the contents of each well were mixed well by pipetting, absorbance was measured at 590 and 630 nm using the Biotek Synergy MX Multi Format Microplate Reader (Biotek, Winooski, VT, USA). The average absorbance at 630 nm was subtracted from the average absorbance at 590 nm, and the relative growth rate was plotted with respect to vehicle control treated cells.

IGROV-1 and SK-OV-3 cells were seeded at the density of 3000 cells in 75 µl media per well in white TC-treated clear-bottom 96-well plates (Costar Cat. No. #3610) and incubated for 24 h at 37 °C, 95% relative humidity, and 5% CO₂. The cells were then treated with vehicle (DMSO) or 10 µM EPZ-5676 followed by immediate addition of Real Time-Glo Annexin V apoptosis and necrosis reagent (Cat. No. # JA1011, Promega Corp., Madison, WI, USA). Luminescence and fluorescence signals were monitored up to 24, 48, and 72 h using a Biotek Synergy MX Multi Format Microplate Reader.

The LDH cytotoxicity assay was performed using the LDH cytotoxicity assay kit from Thermo Fisher Scientific (cat. no. 88953), as previously described. NK92MI cells (1 × 10⁶ cells/ml; 100 μl) served as effector cells and were mixed at a 1:15 ratio in low-attachment round-bottom 96-well tissue culture plates (Costar Cat. No. #7007) with 10 × 10³ IGROV-1 cells that had been pretreated for 48 h with 10 µM vehicle or EPZ-5676. The plates were incubated at 37 °C in a CO₂ incubator for 2 h. After incubation, the 96-well tissue culture plates were centrifuged at 1000 rpm for 3 min. The supernatants were then collected from each well and transferred into a fresh 96-well plate. Then, 50 μl LDH substrate mixture was added to each well. The plate was incubated for 10–20 min at room temperature in the dark, and absorbance at 490 and 680 nm was measured using a Biotek Synergy MX Multi Format Microplate Reader. The absorbance at 680 nm was subtracted from the absorbance at 490 nm to calculate the percent cytotoxicity using the following formula: $\frac{\mathrm{LDH}\mathrm{experimental}-\mathrm{LDH}\mathrm{effector}\mathrm{cells}-\mathrm{LDH}\mathrm{spontaneous}\times 100}{\mathrm{LDH}\mathrm{maximal}-\mathrm{LDH}\mathrm{spontaneous}}$

Melanoma cells (A375, SKMEL-28, M14, and A2058) were seeded at a density of 3 × 10³ cells/well in 75 µL of medium in white TC-treated clear-bottom 96-well plates (Costar, Corning, NY, USA, Cat. No. #3610) and incubated for 24  h at 37 °C, 95% relative humidity, and 5% CO₂. The cells were then treated with vehicle (DMSO) or 10 µM TP-472 for 48 h, followed by immediate addition of Real Time-Glo Annexin V apoptosis reagent (Promega Corp., Madison, WI, USA, Cat. No. # JA1011). Luminescence was monitored using a Biotek Synergy MX Multi-Format Microplate Reader.