Cell viability by LDH and MTT
Cell viability was estimated by Cytotox 96 non-radioactive assay (Promega). The assay was performed according to the Manufacturer’s instructions. Lactate dehydrogenase (LDH) release was assayed after 24 hours of treatment with α-Klotho or LPS by removing 50μl supernatant from each well into a 96-well plate and incubating with cytotox 96 reagents for 30 minutes covered with aluminum foil at room temperature. The stop solution was added, and the absorbance was measured at 490 nm. The percentage of LDH activity was measured by the ratio : (Absorbance of the sample/ Absorbance of maximum activity) x 100%.
The cells were incubated with filtered MTT in DMEM without FBS at 37 °C for 2 hours and 30 minutes. The supernatant was removed from the plate, and DMSO was added. The new supernatant was plated on a 96-well plate and measured at 570nm. MTT % related to control was calculated by the ratio: (absorbance of the sample - absorbance of DMSO)/ (absorbance of the control- absorbance of DMSO) x100%.