Ket6013

Based on our previous findings, we targeted proinflammatory cytokines; chemokine CCL3 (also known as macrophage inflammatory protein 1 alpha), interleukins (IL; IL-6, IL-7, and IL-1β), and tumor necrosis factor alpha (TNF-α) [21 (link)]. The cytokines CCL3 (KET6002; Abbkine Inc.), IL-6 (KET6017; Abbkine, Inc.), IL-7 (MBS453414; MyBioSource), IL-1β (KET6013; Abbkine, Inc.), and TNF-α (ab181421; Abcam, Shanghai, China) were measured in the supernatants of CVF samples via ELISA according to the manufacturer’s instructions. The intra-assay coefficient of variation (CV) was <10% and the inter-assay CV was <12%.

The levels of inflammatory cytokines and chemokines were established in plasma samples and the levels of cortisol and adiponectin were established in serum samples. Undiluted samples were used for inflammatory cytokines and chemokines, and samples for CRP and adiponectin were diluted 200-fold. All biomarkers were analyzed in duplicate using the following enzyme-linked immunosorbent assay (ELISA) kits: IL-1β (KET6013; Abbkine, Wuhan, China), IL-6 (KET6017; Abbkine, Wuhan, China), TNF-α (ADI-900-099; Enzo, Madison Avenue, New York, NY, USA), IL-17 (KET6022; Abbkine, Wuhan, China), CRP (DCRP00; R&D Systems, Minneapolis, MN, USA), adiponectin (DRP300; R&D Systems, Minneapolis, MN, USA), cortisol (ADI-900-071; Enzo, New York, NY, USA), CCL1 (MBS824930; MyBioSource, San Diego, CA, USA), and CCL2 (LS-F146; LSBio, Seattle, WA, USA), according to the manufacturer’s instructions. The standard solutions were respectively prepared using the reagents provided in each kit. Plates were read at 450 nm using a VICTOR X4 Multimode Plate Reader (PerkinElmer, Waltham, MA, USA). Protein quantification was performed using the mean value of the duplicate samples.

Enzyme-linked immunosorbent assays (ELISAs) were used to quantify the levels of Aβ_1–42, IL-1β, IL-6, and TNF-α. To create the total lysate, the cortical tissues of the mice were weighed and homogenized in RIPA buffer containing a cocktail of protease inhibitors (Roche Science, Mannheim, Germany) and a cocktail of phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Brain tissue homogenates were analyzed using the following ELISA kits: Aβ_1–42 (KHB3544, Invitrogen, Waltham, MA, USA), IL-1β (KET6013; Abbkine, Wuhan, China), IL-6 (KET7009; Abbkine, Wuhan, China), and TNF-α (KET7015; Abbkine), according to the manufacturer’s instructions. All the experiments were analyzed in duplicate, and the mean value of the duplicate samples was calculated in all the assays. Quantification of Aβ_1–42 and cytokine levels was performed using a VICTOR X4 Multimode Plate Reader (PerkinElmer, Waltham, MA, USA).