α-Klotho and LPS treatment
On the 15th day, the cells were treated with only DMEM without fetal bovine serum (FBS), LPS, α-Klotho (AA 35-982), or LPS + α-Klotho for 24 hours. A dose-response of recombinant α-Klotho and LPS treatments were made with different concentrations ( α-Klotho 0.1 nM – 4.0 nM) and LPS (0,01µg/mL-100μg/mL) for 24 hours to observe cell viability. After this first screening, only α-Klotho concentrations (0.1 - 2.0 nM) were tested at different time points (1, 4, and 24 hours) to evaluate the α-Klotho effect in LPS (1µg/mL /8 hours) -induced changes in TNF-α levels. Based on these data, the concentration of 1nM of α-Klotho and 1µg/mL to LPS for 4 and 8 hours was used to evaluate changes in NF-kB activity and IL-1β, IL-6 and IFN-γ levels, respectively.
For the experiments involving Glial conditioned Medium (GCM), on the 15th day, cell culture was pretreated with DMEM without FBS in the absence (control) or in the presence of α-Klotho (1 nM) for 24 hours, and then challenged with 1µg/mL LPS for 8 hours. The media were collected and named GCM or GCM-KL. Primary neuronal culture cells were challenged on the 10th day, by changing normal conditioned medium by 25% or 50% of GCM or GCM-KL for 24 hours.