Celltiter glo luminescent assay

Intracellular ATP content was measured in 96 h transfected cardiomyocytes, using the CellTiter-Glo® Luminescent Assay (Promega, Madison, USA), according to the manufacturer's protocol. The ATP content is expressed as luminescence relative units (LRU).

Cellular viability was assessed via ATP content using the CellTiter-Glo Luminescent Assay (Promega, Fitchburg, WI, USA) after a 4-day incubation period, and results represent mean ± standard deviation from three experiments. Total luminescence was measured on a Wallac Multilabel Reader (Perkin-Elmer, Waltham, MA, USA). Cells were treated simultaneously with FRAX1036 (dose range = 0 to 5 μM) or docetaxel (dose range = 0 to 0.4 nM) in an 8 × 10 matrix of concentrations. Combination synergy of FRAX1036 and docetaxel was determined by Bliss independence analyses. A Bliss expectation for a combined response C was calculated by the equation: C = (A + B) - (A × B) where A and B are the fractional growth inhibitions of given doses of drug A and B. ΔBliss scores were summed across the dose matrix to generate a Bliss sum. Bliss sum = 0 indicates that the combination effect is additive while Bliss sum >0 indicates synergy effect and Bliss sum <0 indicates antagonism effect. Statistical analysis comparing the Bliss sums for each cell line was conducted by the Student’s t test.

Cells in suspension were inoculated at an MOI of 1 in 100 µL of appropriate cell culture medium. Eppendorfs with inoculated cells were incubated at +37 °C for 1 h and shaken gently every 10 min. Then, cells were washed with media and seeded in 96-well plates. Wells were first prefilled with 50 µL of cell culture medium, and 50 µL of cell suspension was added on top to obtain a resulting concentration of 5 × 10³ cells per well. Plates corresponding to different experimental time points were infected at the same initial time. Mock-infected cells were included as controls in each plate.
On days 0, 1, 3, 5, and 7, cell proliferation was analyzed with the CellTiter-Glo Luminescent Assay (Promega #G7571, Madison, WA, USA) according to the manufacturer’s instructions. For analysis, 96-well black plates (PerkinElmer #6005660, Waltham, MA, USA) and CellTiter-Glo reagent were equilibrated to room temperature (+22 °C, 30 min). Then, CellTiter-Glo reagent was added to each well, and plates were placed on an orbital shaker (+22 °C, 12 min). Luminescence was quantified on a microplate reader (Promega GloMax Explorer, Madison, WA, USA). All data were normalized to day 0 and expressed as relative cell proliferation.