Elisa detection kits

Quantitation of TNF was performed with ELISA detection kits (Peprotech, NJ, USA) following protocols described by the manufacturer. Briefly, capture antibody at a concentration of 1 μg/mLwas plated on 96-well plates. The plate was sealed and incubated at 37 °C overnight. After the antibody solution was discarded, the plate wells were washed four times with 300 μL wash buffer. Blocking buffer (300 μL) was added to each well, followed by incubation for 1 h at room temperature. The blocking buffer was discarded, and the plate wells were washed four times with 300 μL wash buffer. Saliva (100 μL) was added to each well and the plate was incubated at RT for 2 h. After washing with buffer, 100 μL detection antibody (0.15 μg/mL) was added to each well and the wells were incubated at RT for 2 h. Then streptavidin-HRP conjugates were incubated at RT for 30 min, followed by a color reaction with 100 μL TMB for 20 min. The TNF concentration was determined from a standard curvemade with the TNF peptide available in the kit.

Cytokine levels were measured in cell-free supernatants following 48 h incubation of THP-1 cells, U373 cells, microglial cells, and astrocytes. The cell stimulation protocols in these experiments were the same as that used in protocol 1. Quantitation was performed with ELISA detection kits (Peprotech, NJ) following protocols described by the manufacturer.
For determining TNFα and IL-6 depletion, protocols published in earlier studies were followed [17 (link), 20 (link)]. Briefly, microglia were exposed to LPS/IFNγ, and astrocytes to IFNγ, for 2 days. Their supernatants were transferred to 24-well plates which had been coated with anti-TNFα or anti-IL-6 antibodies (10 mg/ml). After 3-h incubation, the supernatants were transferred to SH-SY5Y cells. After incubation at 37 °C for 3 days, MTT assays were performed on the SH-SY5Y cells.