Quantikine rat tnf α immunoassay

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.

At the end of the experiment (day 11), animals were fasted for 8–12 h before the diet was given, and after 1 h from gavage, blood was collected by portal vein and used for glucose, insulin, TG, HDL-C, low-density lipoprotein (LDL-C), very low-density lipoprotein (VLDL-C), total cholesterol, glutamic oxaloacetic transaminase (SGOT), glutamic pyruvic transaminase (SGPT), gamma-glutamyl transpeptidase (GGT), TNF-α and IL-6 determination. After this, animals were euthanized in a CO₂ chamber. The method used for biochemical parameters measurement was the enzymatic colorimetric (Kit CELM^®, São Paulo, Brazil).
Serum samples were analyzed using commercially available immunoassay kits, according to Vendrame et al. [44 (link)]. Quantikine Rat TNF-α Immunoassay (R&D Systems # RTA00, São Paulo, Brazil) and Quantikine Rat IL-6 Immunoassay (R&D Systems # R6000B, São Paulo, Brazil) were used. According to manufacturer, kit sensitivities were <15 pg/mL and <30 pg/mL for TNF-α and IL-6, respectively.

TNF-α and IGF-I levels were analyzed by enzyme-immunometric using a commercially available kits (Quantikine Rat TNF-α Immunoassay, and Quantikine Mouse/Rat IGF-I Immunoassay, R & D Systems, Minneapolis, MN) and the procedures were performed according to manufacturer’s instructions. All the samples were run in duplicate and the average values are reported.