Prestoblue cell viability reagent

Cell viability was measured using PrestoBlue^® cell viability reagent (Life Technologies). After seven days of maturation, cells were exposed to increasing concentrations of 1,25(OH)₂D₃ (100 nM, 500 nM, 1 µM), anionic UT mix (1-, 2.5-, 5-, or 10-times concentrated) and a combination of 1,25(OH)₂D₃ and UT mix in the previously mentioned concentrations. Following 24 h incubation at 37 °C, 5% (v/v) CO₂, ciPTEC were rinsed once with Hank’s Balanced Salt Solution (HBSS; Gibco, Life Technologies) and incubated with PrestoBlue^® cell viability reagent (diluted 1:10 in complete culture medium), in the dark. After 1 h incubation at 37 °C, 5% (v/v) CO₂, the fluorescence was measured using the Fluoroskan Ascent FL microplate reader, at excitation wavelength of 530 nm and emission wavelength of 590 nm. Data were corrected for the background, normalized to untreated cells, and presented as relative cell viability.

Cell viability was measured with Presto Blue cell viability reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). For this, 5 × 10³ N9 cells were seeded in a 96-well plate 24 h before treatments. Ten microliters of Presto Blue cell viability reagent (Thermo Fisher Scientific, Waltham, MA, USA) were added to the cells at the end of the treatment. The cells were then incubated at 37 °C for 30 min. Finally, the fluorescence was measured at an excitation of 560 nm and emission of 590 nm using a microplate reader (Varioskan Flash, Thermo, Waltham, MA, USA). Relative viability was calculated compared to the untreated control.

RAW264.7 and MET-1 cells were cultured in complete DMEM supplemented with 10% fetal bovine serum (FBS), L-glutamine, and penicillin/streptomycin. Subconfluent cells were incubated for 24 h with increasing concentrations of AuPEI in complete DMEM. For the last 4 h, Presto Blue Cell Viability reagent was added (1/10 final dilution; Presto Blue Cell Viability reagent, A13261; Invitrogen, Waltham, MA, USA). Fluorescence was measured with a 535 nm excitation filter and a 615 nm emission filter. Fluorescence of complete DMEM incubated with Presto Blue was used as background control. Cell viability was calculated according to the formula: $$\mathrm{Viability} \left(\%\right)=\frac{\left[\mathrm{AuPEI} \mathrm{treated} \mathrm{cell} \mathrm{fluorescence}\right]-\left[\mathrm{medium} \mathrm{fluorescence}\right]}{\left[\mathrm{untreated} \mathrm{cell} \mathrm{fluorescence}\right]-\left[\mathrm{medium} \mathrm{fluorescence}\right]} \times 100$$