Alamar blue hs

A549 cells were plated at a density of 1 × 10² cells per well (for 7 days) and at 1.4 × 10² cells per well (3 days). After allowing 24 h for attachment, agents were aliquoted to the wells to reach final concentrations of 10, 5, 2.5, 1.25 and 0.625 µM (1, 0.5, 0.25, 0.125, 0.625 µM for pyrithione) and incubated for 3 and 7 days. Cell viability was assessed by incubating with Alamar Blue HS (Invitrogen A50101). After incubation, 20 µL of Alamar Blue (final concentration 10% v/v) was added to each well and the plates were further incubated for 2 h. Fluorescence intensity was recorded with excitation at 530 nm and emission at 590 nm. These conditions were determined empirically, and this procedure was repeated to generate three biological repeats with a minimum of 6 technical repeats in each set.

Cells were seeded at 500/well in 95 μl in 96-well microplates (Greiner, 655090) and measured every 12 or 24 h by adding alamarBlue HS (Invitrogen) (1/20) and reading in a fluorimeter after 1 hour's incubation, according to the manufacturer's instructions. NM-PP1 or DMSO was added to the cells as noted on the figure. Continuous xCelligence analysis was carried out in 96-well E-Plates (H003957) after seeding the cells at 500/well and NM or DMSO was added as noted on the figure, 36 hours after seeding.

Cells were seeded at 500/well in 95 μl in 96‐well microplates (Greiner, 655090) and measured every 12 or 24 h by adding alamarBlue HS (Invitrogen) (1/20) and reading in a fluorimeter after 1 h's incubation, according to the manufacturer's instructions. 1‐NA‐PP1 or DMSO was added to the cells as noted on the figure.

Cells were incubated for 2 h in Alamar Blue HS (Invitrogen, Massachusetts, U.S.A.) and the fluorescence signal was measured according to the manufacturer’s instructions using the iD3 SpectraMax microplate reader (Molecular Devices, California, USA). For cell growth curves, cells were incubated with Alamar Blue HS at the indicated timepoints.

An Alamar Blue assay to assess cellular viability was performed. Therefore, 5 × 10³ HeLa cells (ATCC, Manassas, USA), each suspended in 100 µL cell culture medium, were seeded on day 0. To prepare the incubation of the cells with Z-OMPD, aliquots of the medium, containing Z-OMPD (0-15 mM) were prepared and adjusted to pH = 7.9, which was the measured pH of the native cell medium outside of the cell incubator, using 1 M NaOH. After 24 h, on day 1 of the experiment, cell medium was exchanged with the medium containing Z-OMPD at concentrations between 0 and 15 mM to start the incubation. After 24 h incubation, on day 2 of the experiment, the Z-OMPD-containing medium was exchanged with the native medium to finalize the incubation. To read out the cell viability, Alamar Blue HS (Invitrogen, Waltham, Massachusetts) was added (10 % in medium per well) and the cells were incubated for three more hours, in order to enable the resazurin to be reduced to resorufin. The fluorescence signal of resorufin was measured with a spectrofluorophotometer (BioTek, Bad Friedrichshall, Germany). Three technical replications of every condition were performed. The experiment was repeated with the cell viability readout happening on day 4 of the experiment (2 days after the end of Z-OMPD incubation).