Victor3 link

MaxiSorp (Nunc, Fisher Scientific) 96-well plates were coated overnight at +4 °C with goat anti-human MANF polyclonal antibody (AF3748, R&D Systems) at 1 μg/ml in 50 mmol/l carbonate coating buffer (35 mmol/l sodium bicarbonate, 15 mmol/l sodium carbonate; pH 9.6). The plate was washed once with phosphate buffered saline, 0.05% Tween 20 (PBST), and incubated with blocking buffer (PBST, 1% casein) at RT for 2 h. After washing with PBST, standard samples of recombinant human MANF (P-101-100, Icosagen, Supplementary Methods) ranging from 62.5 to 2,000 pg/ml and serum samples diluted 1:20 in blocking buffer and pre-incubated on ice for 1 h with 500 mg/l IIR, were added to the plate in duplicate and incubated overnight at +4 °C in agitation (100 rpm). The detection antibody, horseradish peroxidase-conjugated mouse anti-human MANF monoclonal antibody (4E12, Icosagen, Supplementary Methods), was incubated on the plate at 1 μg/ml for 5 h in agitation at RT. Washing with PBST was repeated four times before and after the antibody incubation. Antibodies and samples were applied to the plate in 100 μl volume. For detection, 3,3’,5,5’-tetramethylbenzidine was used according to the manufacturer’s instructions (DuoSet ELISA Development System, R&D Systems). The absorbance was read using a plate reader (VICTOR3 (link), Perkin Elmer) at 450 nm and 540 nm (for wavelength correction).

Cell viability was determined using the MTT assay. For the MTT assay, cells were seeded in a 24-well plate at a density of 2 × 10⁴ cells/ml and incubated for 1 day. Cells were then treated with various concentrations of CFZ or Cis alone, or CFZ + Cis for 24 or 48 h. After incubation, fifty microliter of MTT stock solution (5 mg/ml) was added to each well. After incubation for 2 h, 200 μl DMSO was added to dissolve the formazan precipitates. The completely dissolved formazan was transferred to a 96-well plate. The amount of formazan salts was detected by measuring the optical density (OD) at 570 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader (VICTOR^{3 (link)}, PerkinElmer Life and Analytical Sciences, Turku, Finland). The numerical OD value was quantified as a percentage of the vehicle-treated control.