Msd 5 plex proinflammatory panel 2 rat kit

RBL-2H3 cells were pretreated with various concentrations of AF-343; Ulmus pumila L., Cassia tora L., or Taraxacum officinale extract; or vehicle at 37 °C for 1 h and incubated with compound 48/80 (10 μg/mL) for 24 h. A total of 100 µL of supernatant was used for a highly sensitive multiplex enzyme-linked immunosorbent assay (ELISA) with a Merck Sharp and Dohme Co. (MSD) V-PLEX Proinflammatory Panel 2 (Rat) Kit, which can simultaneously measure IFN-γ, IL-1β, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-13, and TNF-α using an electrochemiluminescence detection method (Meso Scale Diagnostics, LLC., Rockville, MD). The samples and standards were briefly added to 10-spot MULTI-SPOT plates. The lowest limit of detection (LLOD) was calculated according to the manufacturer’s protocol, and the mean value for the plate was used for further calculation of the sample concentrations. Any value below the LLOD for the cytokine assay was replaced with zero in the statistical calculations.

Animals were euthanized using an overdose of sodium pentobarbital (200–300 mg/kg, Vortech; Dearborn, MI) administered by intraperitoneal injection, followed by exsanguination. Weight, nose-to-tail length, and abdominal girth were measured, and body mass index (BMI) was calculated. Blood was collected from the vena cava in EDTA-treated collection tubes and cells were analyzed using a ProCyte Dx Hematology Analyzer (IDEXX Laboratories, Inc.; Norcross, GA). Blood used for serum samples was collected in BD Vacutainer™ Serum Separation Tubes, left at room temperature for 1.5 h, and centrifuged at 25,000 g for 20 min. Fresh serum was used for measurements of high-density lipoprotein (HDL) (#ab65390, Abcam, Cambridge, MA) and blood chemistry (Catalyst Dx Chemistry Analyzer, IDEXX Laboratories, Inc). Epididymal fat pads were removed and weighed. Serum samples were stored at -80 °C until used for ELISA and cytokine analysis. ELISAs were conducted following the manufacturers’ protocols: leptin (#MOB00B, R&D Systems, Minneapolis, MN) and adiponectin (#Acrp30, R&D Systems). Insulin was measured using the Ultra-Sensitive Rat Insulin Elisa Kit (#90060, Crystal Chem; Elk Grove Village, IL). Serum cytokines were measured using the MSD V-PLEX Proinflammatory Panel 2 (rat) kit and MESO QuickPlex SQ 120 (Meso Scale Diagnostics; Rockville, MD).