Genios multiplate reader

The in vitro cell viability was determined using an EZ-Cytox colorimetric cell viability assay kit according to the product instructions (Daeil Lab Service Co. Ltd., Seoul, South Korea). In brief, Daudi cells were seeded at a density of 3 × 10³ cells/well containing fivefold serially diluted IFN-βs (R27T or IFN-β-1a) in a 96-well plate and incubated for 72 h. Reagents were added, and samples were further incubated for 3 h. The optical density was measured at a wavelength of 430 nm using a Tecan GENios Multiplate Reader (Tecan, Raleigh, NC, United States). IC₅₀ values were calculated by non-linear regression analysis using GraphPad Prism 7 (GraphPad Software, San Diego, CA, United States). For the competitive binding assay, Daudi cells were incubated for 72 h with either 1 nM of IFN-βs alone (mock) or with Fc-fusion proteins, and the IC50 values were determined on the basis of the cell viability assay results. Thereafter, the value of the IC50 fold change was calculated by dividing it by the mock value.

Cell viability of microglial cells was determined using the WST-1 Cell Proliferation Reagent (Roche Applied Science, Mannheim, Germany). The assay is based on the cleavage of the tetrazolium salt WST-1 by active mitochondria producing a soluble formazan. This conversion only occurs in viable cells. Cells were incubated with WST-1 for 2 h. Then, the formazan dye formed was quantified by measuring the optical density at 490 nm using a Genios multiplate reader (Tecan, Crailsheim, Germany). The absorbance directly correlated with the number of metabolically active cells.

NO release was quantified by the measurement of nitrite, one of its stable reaction products, in the supernatant of microglial cultures using the Griess reagent. One hundred microliters of the supernatant were mixed with 100 μl Griess reagent [equal volumes of 1% sulfonilamide in 30% acetate and 0.1% N-(1-naphthyl) ethylenediamine in 60% acetate] in a 96-well plate. After 10 min, the optical density at 570 nm was measured with a Genios multiplate reader (Tecan, Crailsheim, Germany). Concentrations were calculated by comparison of absorptions with a standard curve.