Wst 1 reagent

Cell proliferation assays were performed as previously described^{48 (link)}. Cell suspensions of 4 × 10³ cells were plated in 96-well culture dishes for 16 h, and cells were treated with DMSO or different amounts of asteltoxin or CCCP. Two days after treatment, cells were stained with the WST-1 reagent (Merck, NJ, USA), and proliferation was quantified by measuring absorption at 450 nm using an Immuno Mini NJ-2300 (Biotech, Tokyo, Japan).

Cells were harvested directly after isolation and after 24 h stimulation with GM-CSF and adjusted to a final concentration of 1 × 10⁶ cells/mL. Subsequently, 100 µL cell suspension (1 × 10⁵ cells) was transferred to each well of a 96-well culture plate in triplicates. Then, WST-1 reagent ([2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium], Merck, Darmstadt, Germany) was added according to the manufacturer’s instructions followed by incubation for 1.5 h at 37 °C and 5% CO₂. After that, spectrophotometric measurement at an optical density (OD) o440/650 nm was performed on a TECAN microplate reader (Infinite m200, TECAN, Männedorf, Switzerland). The assay is based on converting the tetrazolium salt WST-1 into a colored dye by mitochondrial dehydrogenase enzymes. The results are informative for the metabolic activity of cells and used as an indirect marker for cell viability and linked to inflammatory properties of leukocytes [18 (link)].

Myoblasts were seeded, allowed to differentiate to myotubes, and transfected with AOs at 50 and 12.5 nM as described previously. 24 h after transfection, cytotoxicity was determined by a colorimetric assay using WST-1 reagent (2-(4-iodophenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) (Sigma; cat#: ab65473). Briefly, WST-1 solution was added at a ratio of 1:10 (v/v) per well and incubated for 4 h at 37 °C, 5% CO₂. The absorbance was then measured in a microplate reader (FLUOstar Omega, BMG Labtech) at 450 nm.