Brdu elisa

MLIC assays were performed as previously described.^{27 (link)} Before assay, rat islets were treated for 60 minutes with 50 μg/mL of mitomycin C (Sigma-Aldrich, St. Louis, MO, USA) to suppress islet cell proliferation, and then, plated (30 islets/well) into 96 well plates. Rat splenocytes (2 × 10⁵ cells/well) were then added and the cocultures incubated for 4 days. Phytohemagglutinin (PHA) and splenocytes from other rat strains were used as positive controls. Splenocyte proliferation was assessed by use of a bromodeoxyuridine (BrdU) ELISA (Sigma-Aldrich, St. Louis, MO, USA).

Cell proliferation of LGALS1-deficient ARPE-19 cells was investigated using the 5-bromo-2’-deoxyuridine (BrdU) ELISA (Merck) in accordance with the manufacturer’s recommendations. In brief, 4,000 cells per well were seeded into a 96-well plate and incubated for 24 h under standard conditions to ensure complete cell adherence. Following an additional incubation with BrdU labeling solution for 24 h, cells were fixed and incubated with anti-BrdU antibodies. Following colorimetric development, the amount of BrdU incorporation into the DNA was quantified by absorbance measurement at a wavelength of 450 nm and a reference at 690 nm on the SpectraMax 190 ELISA reader (Molecular Devices, San Jose, CA, USA).
To analyze cell viability, the water-soluble tetrazolium dye (WST-1, Merck) was used in accordance with the manufacturer’s instructions. In brief, 10,000 cells per well were plated into a 96-well plate. Following attachment, cells were cultured in cell culture medium without supplements for 72 h. After an additional incubation in WST-1-containing cell culture medium for up to 2 h, absorbance measurements at a wavelength of 450 nm and a reference at 690 nm were performed on a SpectraMax 190 ELISA reader.