Dy501

For assessing lung inflammation, the level of IL-1β and IL-8, important cytokines in PAH, were measured [22 (link)]. The amounts of IL-1β (Cat number: DY501, R&D Systems) and IL-8 (Cat number: MBS025179, Mybiosource) were measured in the lung homogenates with an enzyme-linked immunosorbent assay. In brief, known concentrations of recombinant rat IL-1β or IL-8 and the experimental samples were added and incubated in polystyrene microtiter plates coated with an antibody against the appointed cytokine, followed by incubation with an enzyme-linked polyclonal antibody directed to the cytokine. Next, a substrate solution for the enzyme was added, and the color development was stopped by adding 2 N H2SO4. The absorbance was measured with a microtiter plate spectrophotometer. The amount of IL-1β, or IL-8 present in each sample was determined from a standard curve generated in each assay and expressed as picograms per milliliter. The sensitivities of the enzyme-linked immunosorbent assays for IL-1β and IL-8 were 5 pg/ml and 2.0 pg/ml, respectively.

Hepatic tissues were homogenized with three volumes of buffer containing 150 mM NaCl, 50 mM Tris-HCl, 0.1% sodium dodecylsulfate (SDS), and 1% Triton X-100. The homogenates were centrifuged at 3000× g and 4 °C for 15 min. Then, the supernatants were collected for the subsequent analysis. Inflammatory cytokines, including IL-1β, IL-6, IL-10, and tumor necrosis factor-α, were detected using commercial enzyme-linked immunosorbent assay kits (dy501, dy506, dy522, and dy510; R&D Systems, Minneapolis, MN, USA).

To examine the levels of proinflammatory cytokines in the striatal lysates, an equal amount of proteins (50 μg) were conducted to the ELISA of proinflammatory cytokines (IL-1β, IL-6, IL-10, and TNF-α) (Cat. DY501, DY506, DY522, and DY510, respectively) (R & D Systems, Minneapolis, MN, the United States according to the manufacturer's instructions (Liew et al., 2019 (link)).