Quantikine rat il 6 immunoassay

IL-6 from coronary perfusate was measured by ELISA method using Quantikine Rat IL-6 Immunoassay (R&D Systems, USA). TnI from coronary effluent was measured by ELISA method using Rat TnI, fast cardiac muscle ELISA kit from Wuhan EIAaB Science Co. (Wuhan, China). Before biochemical analysis all perfusates were concentrated in Amicon Ultra concentrating vessels (EMD Millipore, Billerica, MA, USA). The final volume of concentrate was measured by gravimetry and adjusted to the same final volume for each sample (500 μL).

On the 11th day of the experiment, the animals in the different groups were fasted (8 to 12 h) and subsequently anesthetized (250 mg of tiletamine hydrochloride and 250 mg of zolazepam hydrochloride) for blood collection by cardiac puncture after longitudinal cut made from the base of the abdomen to the outer part, with the aid of surgical scissors. Trained and licensed veterinarians performed all anesthesia, euthanasia, and blood and organ collection procedures.
Then, the blood was collected in tubes with EDTA and destined for the evaluation of plasmatic TNF-α, IL-6, and leptin, being previously centrifuged (3345 RPM/10 min at 4 °C) to separate the plasma, which was submitted to analysis, using the Rat Leptin ELISA kits (Millipore^®—EZRL—83K (Thermo Fisher Scientific, Waltham, MA, USA), Quantikine Rat TNF-α Immunoassay, and Quantikine Rat IL-6 Immunoassay (R&D Systems, São Paulo, Brazil), following the manufacturer’s instructions. All analyses were performed by a professional unaware of the type of treatment received by each animal.
As reference standards of normality, the values obtained from healthy, eutrophic (320–380 g) adult Wistar rats acclimatized in the same conditions of this study and fed with a nutritionally adequate diet (Labina^® diet) were considered to be IL-6 1.74 (0.11) pg/mL, TNF-α 3.26 (0.62) pg/mL, and Leptin 1.76 (0.21) ng/mL.

Blood glucose concentrations for each group were measured immediately following euthanasia using a blood glucose monitor (Medisense, Abbott Laboratories) and reported in mmol/L. All other biochemical measurements were performed using serum prepared from whole blood collected in a serum separator tube and allowed to clot before being centrifuged. Samples were then centrifuged for ten minutes at 14000 rpm and the supernatant removed and stored at −80°C to prevent sample degradation. To assess 4-hydroxynonenal (4-HNE) levels for each animal, serum samples were used in conjunction with a Cell Biolabs’ Oxiselect™ HNE adduct ELISA kit (Catalog Number STA-338). Nitric Oxide (NO) levels were assessed using a NO (total) Detection Kit (Catalog Number ADI-917-020). Assessments of serum interleukin-1β (IL-1β) concentrations were made through the use of a R&D Systems Quantikine Rat IL-1β/IL-1 F2 Immunoassay (Catalog Number RLB00) with serum interleukin-6 (IL-6) concentrations determined through the use of R&D Systems Quantikine Rat IL-6 Immunoassay (Catalog Number R6000B).