Earlytox cardiotoxicity kit

In this technique, intracellular calcium fluxes are visualized by fluorescent reporter dyes, ratiometric calcium indicators like the Fura dyes or nonratiometric dyes such as the fluorescent dyes. All these indicators have slight adverse effects on cell physiology, but we chose the EarlyTox Cardiotoxicity Kit from Molecular Devices (San-Jose, California, USA), which was reported to be less toxic than the conventional dyes.
1 × 10⁵ HL-1 cells were seeded per well of 96-well plate 24 hours prior to the exposure. Samples in different dilutions and positive control (40-µM propranolol hydrochloride, VWR, Radnor, Pennsylvania, USA) and EarlyTox Cardiotoxicity Kit (Molecular Devices, San-Jose, California, USA) were added for 2 hours at 37°C according to the manufacturer’s protocol.^{30 (link)} Spontaneous calcium transients were captured in single cells at Ex 470 nm/Em 500-530 nm for 30 seconds using a Nikon high content screening system (Nikon CEE GmbH) prior and after the addition of samples and positive control. After subtraction of the background, the number of spikes (only those with amplitudes >30 AFU) per 30 seconds was manually counted before and after the treatment.³¹ Testing was performed in triplicates.

Calcium flux in cardiomyocytes was assessed using the instructions provided by the EarlyTox Cardiotoxicity Kit from Molecular Devices, LLC (Table 1). Briefly, cells in 25 μL medium per well were equilibrated for 2 h at 37°C in the presence of 25 μL of prewarmed calcium dye reagent. Before treatment with test chemicals, basal calcium flux was recorded at 515–575 nm following excitation at 470–495 nm for 100 s in 0.125 s read intervals using the FLIPR tetra plate reader (Molecular Devices). The exposure time per read was 0.05 s, the gain was set to 2,000, and the excitation intensity was set to 30%. The instrument temperature was kept at a constant 37°C. Test chemicals (12.5 μL of 5× concentration working solutions) at the appropriate concentrations were added simultaneously to all wells using the instrument-specific automated liquid handler. Between subsequent readings at 10, 30, 60, and 90 min, cells were incubated at 37°C and 5% CO₂.