Alamarblue assay

GBM8 cells were seeded at 2500 cells per well in 96-well plates. The next day increasing concentrations of inhibitor was added. After 4 days neurospheres were viewed and photographed under a Nikon microscope (4 × objective). Cell viability was determined by Alamar Blue assay after 8 days (Thermo Fisher Scientific).
For cell viability in high or low glucose, glioma cells were seeded in 96-well plates (2500 cells per well). The following day cells were washed twice with PBS, and glucose free DMEM supplemented (DMEM 1X, with l-Glutamine, without d-Glucose, without sodium pyruvate) (Gibco by Life Technologies, cat#11966-025) with 1% dialyzed FBS (Gibco by Life Technologies, cat#26400-036), penicillin–streptomycin (Corning), and d-(+)-Glucose solution (SIGMA), to either 2 or 25 mM final concentration, was added. Increasing inhibitor concentrations were added and incubated with cells for 72 h. Cell viability was determined by Alamar Blue assay (Thermo Fisher Scientific).

The cytotoxicity of AX films was investigated by performing alamarBlue® assay (Invitrogen, Carlsbad, CA, USA) on baby hamster kidney cells (BHK-21, ATCC^®, Manassas, VA, USA) using an indirect contact method (according to ISO-10993 standard) [13 (link)]. The culture medium was prepared by adding 10% fetal bovine serum (FBS) and 1% antibiotic (penicillin-streptomycin) in Dulbecco’s modifed Eagle’s medium (DMEM). The cells were incubated in CO₂ atmosphere at 5% RH and 37 °C to achieve 80% confluence. The AX films (50 mg pieces) were sterilized by UV light (45 mins for each side) and then incubated with culture medium for 24 h to obtain extracts of films. The cells were suspended in culture media, and approximately 1 × 104 cells were seeded in each well of 96-well cell culture plates. The plates were incubated for 24 h, and then medium was replaced with 100 µL AX films extracts in sample wells and fresh medium in control wells. Then plates were again incubated for 24 h, and alamarBlue® assay (Invitrogen, Carlsbad, CA, USA) was performed to estimate the percentage of viable cells by measuring the optical density of the control and sample wells using microplate reader at 570 nm [33 (link)].

Cell viability assay was done using the alamarBlue assay according to the manufacturer's recommendations (Thermo Fisher Scientific) where cells were cultured in 96-well plates in 200 μl of the medium. On day10, 20 μl/well (10%) of alamarBlue substrate was added and plates were incubated for 1 hr in the dark at 37°C. Readings were taken using BioTek Synergy II microplate reader (BioTek Inc., Winooski, VT, US) using fluorescent mode (Ex 530 nm/Em 590 nm).

Cell viability was assessed using the AlamarBlue assay (Thermo Scientific), according to the manufacturer’s protocol. Upon entering the viable cell, resazurin–the active compund of AlamarBlue–is reduced to resorufin that is assessed by fluorescence at Ex₅₆₀ nm/Em₅₉₀ nm.
Furthermore, cell viability was assessed by a live/dead fluorescence-based cell assay using fluorescein diacetate (FDA) and propidium iodide (PI). Staining of cells was performed using a mixture of 0.5 μg/ml FDA (Sigma-Aldrich) and (0.05 μg/ml PI), (Sigma-Aldrich) in PBS for 30 s. Cells treated with 0.5% Triton X-100 (Sigma-Aldrich) for 2 min served as a positive control for cell death. Following staining, cells were washed with PBS and examined by fluorescence microscopy. Image J v2.0 software was employed for quantification. Fluorescence images were converted into single-channel 8-bit grayscale images, and the threshold was adjusted to measure the mean gray values. FDA mean fluorescence intensity was divided by the corresponding PI mean fluorescence intensity to calculate the FDA/PI ratio at day 0 and day 7.

The caspase-3/-7 activity was measured using the Caspase-Glo^® 3/7 assay according to the manufacturer’s instructions (#G8090, Promega AG). Cells were treated in a six-well format. At each timepoint of the assay, 7.5 × 10⁴ cells/well were transferred to a 384-well plate and assessed for their Caspase-3/-7 activity.
Caspase activity was inhibited using pan-caspase inhibitor Q-VD-OPh (#S7311, Selleckchem/Lubio Science, Zurich, Switzerland) at indicated concentrations. Q-VD-OPh was added at the beginning of the experiment and was replenished once after 48 h.
To assess relative viability upon ATRA treatment, the alamarBlue^® assay was used (#DAL1025, Thermo Fisher). Cells were cultured in 96-well plates for the duration of the assay and 10% alamarBlue^® was added at the end. Absorption was measured after 3 h.
When measuring viability upon nutrient restriction, CellTiter-Glo^® luminescent cell viability assay (#G7570, Promega AG) was used to assess viability dependent on ATP availability. The assay was performed according to the manufacturer’s protocol in a 384-well format using 12.5 × 10⁴ cells/well.

Cells were seeded into 24-well plates (Falcon, BD Biosciences, San Jose, CA, USA) at a concentration of 1 × 10⁵ cells/well and incubated for 24 h at 37 °C in 5% CO₂ atmosphere. Cells were treated with NBP-J for 10, 30, 60, and 180 s, followed by an additional incubation. Cell viability was evaluated using the Alamar blue assay, a redox fluorogenic symbol of metabolic reduction (Fischer Scientific, Ballycoolin, Ireland). The control group without plasma treatment was evaluated by all assays. All assays were performed using three independent sets of tests.