Wst 1

The cell viability was evaluated after incubation with different concentrations of CB[6] and CB[7] (1, 0.5, and 0.1 mM) using the WST-1 method. PBMCs were seeded at 10⁵ cells/well into a flat-bottomed, 96-well plate. After 72 h of cultivation, 10 μL of WST-1 (Takara Bio, Kusatsu, Japan) stock solution was added into each well containing 100 μL of cell suspension. The absorbance was directly read at 450 nm, and the reference was read at 620 nm.

Cell viability following the exposure to MIPs followed the ISO-10993 standard [20 ]. Briefly, L929 cells (2000 cells/well) were seeded on a 96-well culture dish for 24 h. The extraction solution (0.2 g/mL) were prepared by PBS for 24 h at 37 °C. After 24 h, the L929 culture dish removed supernatants. Then, 100 μL of the experiment extracts solution (the extraction solution and fresh medium were mixed in the ratio of 1:9) was added into a 96-wells culture dish and incubated for 24 h. After that, 100 μL of WST-1 (water-soluble tetrazolium salt; Takara, Japan) solution was added to each well and incubated for 1 h. Using an ELISA reader, the absorbance values of each well were measured at the wavelength of 450 nm.

Cell proliferation was examined by WST-1 (Takara Bio, Mountain View, CA, USA). The specific method is stated in our previous study [29 (link)].

The A9 stable cell line in which ELuc was expressed under the control of CAG promoter was seeded in a 96-well clear bottom plate (Nunc, Wiesbaden, Germany) at the density of 1 × 10⁴ cells per well. After 1 day, the medium was replaced with DMEM without phenol red (Gibco-BRL, Grand Island, NY) but supplemented with 10% FBS, 25 mM Hepes/NaOH (pH 7.0, Sigma-Aldrich), and 200 μM D-luciferin (Toyobo), and incubated for 48 h in the presence of various concentrations of SDS (Nacalai Tesque). Bioluminescence was nondestructively measured for 5 s in each well at 37 °C using a microplate-type luminometer (AB-2350 Phelios, ATTO, Tokyo, Japan), and then viability of the same cells was measured using the cell proliferation reagent WST-1 (Takara, Shiga, Japan) according to the manufacturer’s protocol. Absorption was measured at 440 nm using a microplate reader Infinite F2000 PRO (Tecan, Männedorf, Switzerland).

The cytotoxicity of suramin sodium salt on Vero cells was measured using the water soluble tetrazolium salt-1 (WST-1) assay (Takara Bio Inc., Kusatsu, Shiga, Japan) following the manufacturer’s protocol. Briefly, Vero cells were treated with several dose of each drug (5, 10, 50, 10², 10³, 10⁴µM) for 48 h, and then incubated with premix WST-1 for 1 h at 37 °C. Absorbance was measured at 430 nm using ELISA plate reader (Epoch, Bio-Tek Instruments, Inc., Winooski, VT, USA). The 50% cytotoxic concentration for inhibitors (CC₅₀; drug concentration that reduced the cell viability by 50% compared to the cell only control) was determined using nonlinear regression analysis (GraphPad Prism 8.4). Experiments were performed in duplicate wells and repeated three times.

The cytotoxic effects of TMZ and CLD were determined by using the cell viability assay reagent WST-1 (Takara Bio, Japan), as described previously [21 (link)]. Briefly, glioma cells were seeded at the density of 1–1.5 × 10³ cells/well in 96-well flat-bottomed plates and incubated at 37 °C overnight. Afterward, the cells were treated with TMZ (0, 62.5, 125, 250, or 500 μM) or CLD (0, 110, 220, 440, or 660 μM) for 72 h. Following treatment, WST-1 reagent (10 μL) was added to each well and incubated for 1–2 h at 37 °C. Absorbance was measured at 450 nm using a microplate reader. The viability of untreated cells was considered as 100%.

Cell proliferation assays were performed using WST-1 (Takara, Otsu, Japan) according to the manufacturer's instruction. Briefly, B16F1 or B16F10 cells (1 × 10⁴ per well) were plated in 96-well microplates for one day and then treated with IMQ after pretreatment with 3-MA (10 mM). At each time point, the WST-1 reagent was added to the 96-well plates, and the plates were then incubated for 4 h. The absorbances at 450 nm and/or 690 nm were measured using a plate reader (Biotek Instruments Inc., Winoski, VT, USA).