Assays for Screening Safe Concentrations and Effects of DIO on Normal DAKIKI Cells

To begin, seed DAKIKI cells in 96-well plates at a density of 0.4 million cells per well. Set up the different experimental groups, and after, the corresponding treatments based on the grouping method. Incubate the plates for 24 hours at 37 degrees Celsius and 5%carbon dioxide.

For the lactate dehydrogenase cytotoxicity assay, add five microliters of lysate to each well in the high control group. And incubate the plate at 5%carbon dioxide and 37 degrees Celsius for 15 minutes. Then, add 100 microliters of the reaction mixture to each well.

To quench the reaction, add 50 microliters of the stop reaction solution per well. For the Cell Counting Kit-8, or CCK-8, assay, after the initial 24-hour incubation, add 20 microliters of CCK-8 reagent to each well. And return the plates to the incubator at 5%carbon dioxide and 37 degrees Celsius for two hours.

The results of the lactate dehydrogenase cytotoxicity assay showed insignificant cytotoxicity induced by dioscin at concentrations of 0.25 to 1.0 micrograms per milliliter, which had lactate dehydrogenase release rates below 10%CCK-8 assay showed that, compared with the model group, dioscin inhibited LPS-induced DAKIKI cell proliferation in a concentration-dependent manner, with the concentrations of 0.5 and 1.0 micrograms per milliliter showing significant inhibition.