Celltiter glo 2.0 viability assay

For 96-well hiPSC-CM differentiation assays, hiPSCs were plated in Matrigel-coated 96-well plates at 1000 cells per well and allowed to adhere for 4 days. The hiPSCs were subsequently treated with bioactive lipids at the indicated concentrations and durations and assessed following day 8 of the chemically-defined hiPSC-CM differentiation protocol. Immunostaining using previously-published protocols was conducted to qualitatively assess cell viability and cardiomyocyte differentiation efficiency^{45 (link)}. Fluorescence intensity and cell number was quantified using ImageJ software. For quantitative viability measurements, cells were treated with CellTiter-Glo 2.0 Viability Assay (Promega) or PrestoBlue reagent (Life Technologies) per manufacturer-recommended procedures. 96-well imaging and viability assays were conducted using a Cytation 5 plate reader/imager (BioTek Instruments). Prism (GraphPad) was utilized for graph generation and statistical analysis. Confocal imaging was performed using a Zeiss LSM 510Meta microscope (Carl Zeiss) using Zen software.

In a similar fashion to the protocols mentioned above in Section 2.2, we plated cells in a standard 96-well plate at a cell density of 3000 cells/100 μL and incubated for 24 h. We diluted artesunate, carboplatin, and paclitaxel stock solutions with DMSO and media to achieve a final concentration of 40 μM, 16 μM, and 32 μM, respectively. Each drug was added, as indicated, for 24 h and treatment media were then replaced with fresh media. Drug administration sequences were artesunate on day 1 (D1A) or day 2 (D2A), carboplatin and paclitaxel on day 2 (D2C/T), carboplatin, paclitaxel, and artesunate on day 2 (D2C/T/A), or artesunate on day 1 followed by carboplatin and paclitaxel on day 2 (D1A; D2C/T). Cells were incubated at standard growth conditions for a total of 72 h. Viability measurements were determined using the CellTiter-Glo 2.0 viability assay (Promega). Luminescence was measured using a Varioskan LUX multimode microplate reader (ThermoFisher Scientific). Statistical analysis was performed using GraphPad Prism (v5.01).

Tissue culture wells (96-well plates) were coated with recombinant human PECAM-1/CD31, P-selectin, E-selectin and CD14 (non-adhesion control) human IgG1 Fc fusion proteins (purchased from R&D Systems) at a concentration of 5 μg/mL in 30 μL of Tris–HCl 20 mM pH 8.6, overnight at 4°C. Non-coated control wells were also included. The next day, unbound proteins were removed and the wells were washed with PBS before being blocked for one hour in X-VIVO media at 37°C. Ten thousand KG1a cells were then added into each well, in a volume of 100 μL of X-VIVO. The plate was incubated for 7 h at 37°C before Ara-C or saline control was added in 20 μL at final concentration of 10 μg/mL, 100 μg/mL, or 1 mg/mL. After 48 h of incubation at 37°C, cell viability was assessed using the CellTox Green Cytotoxicity assay and CellTiter-Glo 2.0 viability assay (both from Promega) on a PHERAstar microplate reader (BMG Labtech) according to the manufacturer’s instructions.
Two negative (non-adhesion) controls were included for chemo-sensitivity assay, (1) parallel blocked non-coated wells, and (2) parallel control wells coated with recombinant human CD14 IgG1 Fc fusion protein (as non-adhesion control).

Cells were seeded in white-walled 96-well microplates at 3x10³ cells per well in 100 μL growth media and incubated for 24 hours at 37°C, 5% CO₂ to allow cells to attach. Subsequently, the growth media was removed and replaced with fresh media containing serially diluted drug(s) of interest or blank media for untreated controls. Within an experiment, each drug concentration was tested in duplicate. For paclitaxel, drug concentrations ranged from 3000 nM to 0.017 nM. Lapatinib concentrations spanned from 50 μM to 0.08 μM. After treatment, we incubated cells an additional 96 hours, then determined cell viability of drug-treated cells relative to untreated control cells (% viability) using the CellTiter-Glo 2.0 viability assay (Promega). Luminescence was measured using a Varioskan LUX multimode microplate reader (ThermoFisher Scientific). Dose-response curves were then fit to the data (four parameter log-logistic model), and we calculated IC₅₀ values with R statistical software (version 3.5.0), package drc (version 3.0) [44 (link)]. Each drug was analyzed with each cell line using data from a minimum of three independent experiments.