Brdu proliferation assay kit

HMECs (1×10⁵ cells/mL) were cultured in serumfree media supplemented with
glucose at two different concentrations, in 96-well microplates for 24 hours. Cells were
then incubated with treatments in the presence of bromodeoxyuridine (BrdU) at a final
concentration of 0.01 mM for another 24 hours. Cells were then fixed and incubated with
anti-BrdU antibody for 90 minutes. Detection was performed using the colorimetric BrdU
Proliferation Assay kit (Roche, Germany), according to the manufacturer’s instructions.
Optical density was measured at 450 and 650 nm and the results are expressed as percentage
of the control.

BrdU proliferation assay kit was purchased from Roche Applied Science (Mannheim, Germany) and used according to the manufacturer’s protocol.

hCECs (1 × 10⁴) were cultured in 96-well plates and treated with miRNA or IFN-γ for 48 h. Cell viability was measured using the Cell Counting kit-8 (CCK-8; Dojindo, Kumamoto, Japan). The plates were incubated with CCK-8 solution for 1–2 h. Cell viability was determined by measuring the absorbance at 450 nm using a microplate spectrophotometer (Synergy HTX, BioTek, Winooski, VT, USA).
The cell proliferation rate was measured using a commercial bromodeoxyuridine (BrdU) proliferation assay kit (GmbH; Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s protocol. Cells (5 × 10³ cells/well) were seeded into 96-well plates. Cells were labeled with BrdU at 37 °C and 5% carbon dioxide (CO₂) for 48 h. After incubating the plate in the FixDent solution at 25 °C for 30 min, the cells were incubated with the anti-BrdU-POD solution at 25 °C for approximately 90 min. Then, the substrate solution was added to each well and the plate was incubated at 25 °C for 20 min. Thereafter, 1 M sulfuric acid (H₂SO₄) was added to each well to stop the reaction. Optical density was measured at 450 nm using a microplate reader (Synergy HTX, BioTek). Proliferation rates were expressed as the percentage of controls after subtraction of the corresponding blanks.

Peripheral blood mononuclear cells (PBMCs) were isolated from sterile heparinized blood by Ficoll/Hypaque gradient centrifugation. PBMCs were washed twice with RPMI-1640 (catalog 11875093, Thermo Fisher Scientific), counted, and stimulated with phytohemagglutinin (catalog 11249738001, Roche), bacillus Calmette–Guérin, and candida. The negative control contained the cells without any stimulation. The cultures were performed in duplicate and incubated for 6 days in a humidified 37°C incubator with 5% CO₂. BrdU proliferation assay kit (Roche) was used to measure cell proliferation. The stimulation index was calculated as the mean ratio of the OD of stimulated cells divided by the OD of unstimulated cells.