Blood samples were centrifuged for 10 min at 3000 rpm and the resulting serum was stored at −80 °C. The cytokines IL-6 and of TNF-α were determined using Rat DuoSet ELISA kits (R&D Systems; Minneapolis, MN, USA) following the instructions supplied by the manufacturer. The limit of detection of these kits was 125 pg/ml for IL-6 and 62.5 pg/ml for TNF-α.
Rat duoset elisa kits
Manufactured by R&D Systems
Sourced in United States
Rat DuoSet ELISA kits are a set of reagents designed for the quantitative measurement of rat-specific proteins or other analytes in biological samples. The kits provide the necessary components for the development of sandwich ELISAs, including a capture antibody, a detection antibody, and a calibrator.
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3 protocols using rat duoset elisa kits
1
Cytokine Measurement in Rat Serum
2
Cytokine Profiling in Placental Tissues
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Placental tissues were weighed and placed in a Biomasher II homogenizer (Kimble Chase). One milliliter of lysis buffer (T-PER protein extraction reagent containing HALT protease inhibitor cocktail with EDTA according to the manufacturer's instructions; Pierce Biotechnology, Inc.) per 100 mg of tissue was added and the placental tissue was mechanically homogenized. Tissue homogenates were centrifuged at 10 000 × g at 4°C and supernatants were transferred to clean tubes and frozen at –20°C prior to analysis. Placental protein extracts, amniotic fluids, and serum from dams were diluted 1:5 in the Reagent Diluent (R&D Systems) and analyzed for the presence of interferon gamma (IFNG), tumor necrosis factor (TNF), interleukin (IL6), and IL10 using rat DuoSet ELISA kits (R&D Systems). Cytokine values were expressed as pg/mg placental tissue or pg/ml amniotic fluid or serum. Standard curves were constructed using recombinant cytokines provided in the kit, and these curves were included for each assay. For placental homogenates, the recombinant proteins were diluted in Reagent Diluent:T-PER (4:1 vol/vol).
3
Cytokine Quantification in Brain Injury
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An additional cohort of animals underwent the procedures mentioned above (Figure 6; C+N = 3; C+PostC = 3; HI+N = 11; HI+PostC = 12; males = 15; females = 14). To measure cytokines prior to returning to basal levels, 5 d after injury, pups were euthanized with pentobarbitone sodium (100 mg/kg, i.p.) and brains were removed. Cortical tissue was homogenized in ice cold lysis buffer (20 mmol/l Tris-HCl (pH 7.5); 150 mmol/l NaCl; 1 mmol/l EDTA; 0.5% Triton X-100; 0.1% SDS) and centrifuged at 5,000 rpm × 2 min at 4 °C to remove cellular debris. Total protein concentration was determined using a commercial protein assay kit (Bio-Rad DC protein assay, Hercules, CA). Concentrations of IL-1β and TNF-α were determined using rat duoset ELISA kits (from 100 µg of protein per sample, in triplicate) according to the manufacturer's instructions (R&D systems, Minneapolis, MN). The absorbance values were read on a FLUROstar Optima plate reader (BMG LabTek, Ortenberg, Germany).
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