Click it edu microplate assay kit

NP cell proliferation was evaluated by CCK-8 assay and EdU incorporation assay. NP cells were seeded in the six-well plate (2 × 10³ cells/well) and incubated for 3 or 7 days in different acidic culture media. Then, one group of NP cells were used to perform the CCK-8 assay according to the manufacturer’s instructions (Beyotime, China). Another group of NP cells were used to measure EdU incorporation ability using a Click-iT EdU microplate assay kit (Invitrogen, U.S.A.). In these two assays, NP cell proliferation potency was expressed as the absorbance at 450 nm wavelength and as the relative fluorescence units (RFUs) detected at an excitation/emission wavelength of 490/585 nm, respectively.

NP cell proliferation was detected with a Cell Counting Kit-8 (CCK-8, Beyotime, China). Briefly, 3 × 103 cells/well were seeded in a 48-well plate and incubated with medium containing different test compounds for 1, 3 and 5 days after which they were incubated with CCK-8 solution for 2 hours at 37 °C. Then, 200 μL supernatant was used to measure the potency of cell proliferation as indicated by the absorbance at a wavelength of 450 nm. NP cell proliferation was also evaluated by the uptake of 5-ethynyl-2′-deoxyuridine (EdU) into DNA, using a Click-iT EdU microplate assay kit (Invitrogen, USA) according to the manufacturer’s instructions. Briefly, after NP cells (seeded in a 96-well plate, 1.5 × 10³ cells/well) were incubated with different test compounds for 1, 3 and 5 days, NP cells were labeled with EdU which was coupled to Oregon Green-azide. Then, EdU incorporated into cell DNA was detected using an HRP-conjugated anti-Oregon Green antibody and Amplex UltraRed. The NP cell proliferation rate was detected at an excitation/emission wavelength of 490/585 nm using an automatic microplate reader (Thermo, USA) and was expressed in relative fluorescence units (RFU).