Synergy 4 microplate reader

Cells were grown on U-bottom 96-well plates at respective concentrations sufficient to keep untreated cells under exponential growth by the time of read-out after treatment. Cells were exposed to each drug alone and to the combination of both drugs on the same plate in 48 h experiments. In sequential experiments, compounds were added 48 h apart in 72 h experiments. Cell viability was then determined using a fluorometric assay based on the resazurin reduction activity of the cells (Cell Titer Blue, Promega) and confirmed by trypan blue dye-exclusion (Sigma). Fluorescence was determined for controls and three replicates per treatment condition using the Synergy4 microplate reader (BioTek). Standard curves were obtained for each individual cell line with the cell count and fluorescence values. The number of viable cells was obtained using the least-squares regression method of the standard curve and by doing a ratiometric quantification of viable cells normalized to the respective controls. Experiments were conducted in triplicates. A cell killing effect was calculated as the 1 — normalized viability value. Dose necessary for 50% of growth inhibition (GI₅₀), combinatorial indexes (CI) and Dose Reduction Indexes (DRI) for a fraction affected (Fa) of 0.5, were determined using the CompuSyn software (Biosoft).

The ADCC Reporter Bioassay Kit (Promega Life Sciences) was used to measure the induction of ADCC by serum antibodies. The 293T cells were seeded on poly-D lysine coated, flat bottom white 96-well tissue culture plates (Costar) at 50,000 cells per well the day before transfection. Twenty-four hours later, cells were transfected with pCAGGS PR8 M2 (200 ng/well). The next day, transfected cells were washed with 100 µL of PBS and supplemented with 25 µL of Roswell Park Memorial Institute (RPMI) medium (Thermo Fisher Scientific). Pooled mouse sera were diluted 1:3 (from a starting dilution of 1:30) in RPMI medium and added (25 µL per well) to the transfected cells in triplicates. ADCC mouse effector cells (Promega Life Sciences) were added at a concentration of 75,000 cells per well and incubated for 6 h at 37 °C. At the end of the incubation, 75 µL of Bio-Glo luciferase assay substrate (Promega Life Sciences) was added to each well and incubated at RT for 5 min. Luminescence was read using a Synergy 4 microplate reader (BioTek) and Gen5 2.09 software. Fold induction over baseline was calculated and graphed using Prism 7.0.

According to the previously reported method (Yang et al., 2019 (link)), the human pancreatic cancer cell line (Panc 04.03) was seeded at the density of 2.4 × 10⁴ on 96-well plates. After incubation at 37°C with 5% CO₂ for 24 h, the cells were incubated with different concentrations of inhibitors under the same conditions. After 72 h, MTT (0.5 mg/ml, 100 μl) was added after the supernatant was discarded. Then keep the 96-well plate at 37°C for 4 h. After that, abandon the medium and inject 200 μl of dimethyl sulfoxide (DMSO) into each well. The spectrophotometric absorbance of the sample at 570 nm was measured using a Synergy 4 Microplate Reader (BioTek Instruments Inc., United States). All of the compounds were tested three times in each of the cells.

The activity levels of single agents and combinations were determined by a high-throughput CellTiter-Glo cell viability assay (Promega). Cells (1–2 × 10³) were plated in each well of 384–well plates using a Precision XS liquid handling station (Bio-Tek Instruments, Winooski, VT) and incubated overnight. Drug source plates were prepared in 96-well Megatiter plates (Neptune Scientific, San Diego, CA), and the Precision XS station was used to transfer drugs to four replicate wells with an additional four control wells receiving DMSO vehicle control without drug. At the end of the drug incubation period, CellTiter-Glo or Caspase-Glo reagent was added to each well at 1:1 ratio (v/v) with media. The luminescence of the product of viable cells was measured with a Synergy 4 microplate reader (Bio-Tek Instruments). The luminescence data were transferred to Microsoft Excel to calculate percent viability. IC50 values were determined using a sigmoidal equilibrium model regression and XLfit version 5.2 (ID Business Solutions). The IC50 values obtained from single-drug cell viability assays were used to design subsequent drug combination experiments. High-throughput two-agent combination screening experiments were performed using a 5 × 5 matrix format in 384-well plates to interrogate 25 individual concentration ratios per combination (Supplemental Figure S1).

Measurement of [Ca²⁺]_i was performed on SMCs from 2 to 3 passages and on HUVECs from 2 to 4 passages grown in 48- or 96-well plates. The cells were loaded with 2 µM Fura-2/AM during one-hour incubation at 25 °C in a physiological salt solution (PSS) containing (in mM) NaCl (145), KCl (5), MgCl₂ (1), CaCl₂ (1), Hepes (5), and d-glucose (10) at pH 7.4. Pluronic F-127 (0.02%) was used to facilitate Fura-2/AM loading. Registration of the calcium signal was done in the cells incubated in PSS at 25 °C. The fluorescence was measured in parallel from three to six wells at excitation wavelengths of 340 and 380 nm and emission wavelength of 505 nm, using a Synergy 4 Microplate Reader (BioTek, Winooski, VT, USA). The increments in [Ca²⁺]_i are presented as ratios of the fluorescence at 340 and 380 nm.