Elisa microplate reader

The IL-1α and TNF-α are separately used for the anti-inflammatory capacity. The NIH/3T3 cells are cultured in 96-well plates (5 × 103 cells/mL) for twenty-four hours in a carbon dioxide incubator. The culture solution is removed and then replaced with another culture solution in which the samples were once immersed, after which the cells are once again cultured in the carbon dioxide incubator for twenty-four and seventy-two hours. The cells in the 96-well plates are removed in order to remove 50 µL of supernatant fluid. The fluid is added to other 96-well plates, followed by 50 µL of antibody cocktail. Afterwards, the mixtures are oscillated at 400 rpm for one hour, and the solution is removed. After the plates are rinsed with wash buffer and 100 µL of TMB development solution is added, the plates are oscillated at 400 rpm in darkness for ten minutes. Finally, 100 µL of stop solution is added for interaction, and then a micro plate ELISA Reader (Tecan, Sunrise, Zurich, Switzerland) is used for the measurement with a specified OD of 450.

The cytostatic effect of metapristone was determined by the 3-[4, 5- dimethylthiazol]-2, 5-diphenyltetrazolium bromide (MTT) assay as previously described [45 (link)]. Cells (A549 and H1975) were plated in 96-well plates and treated with 5-80 μM concentrations of metapristone for 24 h, 48 h and 72 h, respectively. After incubation for specified times at 37 °C in a humidified chamber, MTT reagent (5 mg/mL in PBS) was added to each well and incubated for another 4 h. The MTT solution was removed from the wells by aspiration and the formazan crystals were dissolved in DMSO (150 μL). Absorbance was detected on a microplate ELISA reader (Tecan, Switzerland) at 570 nm. The concentration of metapristone which gives a 50% growth inhibition value was defined as the IC₅₀.

NGF-induced differentiated PC12 cells were seeded in differentiating medium at 10 × 10³ cells/well in a 96-well microplate for assessing SCO and CTFE cytotoxicity by the WST-1-based method. After exposure to different concentrations of CTFE (0, 50, 100, 200, and 500 µg/mL) and SCO (0.0, 0.5, 1.0, 2.5, and 5.0 µg/mL) for 24 h, the cells were incubated with 110 µL of differentiating medium containing 10% EZ-Cytox Reagent (Daeil Lab Service, Seoul, Korea) at 37 °C for 2 h. Afterward, absorbance was measured at 450 nm using a microplate ELISA reader (TECAN, Männedorf, Switzerland).

To assess the effect of CTFE on AChE, the differentiated PC12 cells were seeded in differentiating medium at 10⁴ cells/well in a 96-well microplate for assaying the AChE activity. The cells were exposed to 2.5 µg/mL SCO and different concentrations of CTFE (50, 100, and 200 µg/mL) for 24 h. Afterward, the AChE activity was measured at 37 °C for 10–30 min using an AChE Colorimetric Assay Kit (BioVision, Mountain View, CA, USA). Absorbance at 570 nm was measured in a microplate ELISA reader (TECAN). The effects of SCO and CTFE on AChE activity were determined by comparing the absorbance values with the standard curve of AChE.