Elisa assay kit

Conscious BP was measured as detailed previously (Javkhedkar et al. 2012 (link)). Briefly, 5–6 weeks old rats were anesthetized with isoflurane and a midline abdominal incision was made to expose the aorta. The catheter of the radio transmitter was inserted into the abdominal aorta, guided upstream, and secured with tissue adhesive. The body of the telemetric device was placed in the abdominal cavity and sutured to the abdominal musculature. Analgesia (buprenorphine, 0.05 mg/kg SQ) was administered every 12 h for 2 days and animals were allowed to recover for 1 week prior to the recording of BP. Urinary 8-isoprostane was measured by RIA kit (Cayman, Ann Arbor, MI) and nitrotyrosine was determined by using an ELISA assay kit (Millipore, Billerica, MA). Malondialdehyde was measured by the method of Mihara and Uchiyama (Mihara and Uchiyama 1978 (link)).

Mice were deprived of food overnight, and serum was collected. The serum cytokine content was measured using a milliplex enzyme-linked immunosorbent assay (ELISA) assay kit (Millipore, Billerica, MA, USA). Serum FFA levels were also measured with an ELISA kit (Cusabio, Wuhan, China). Serum levels of fasting glucose, TG, AST and ALT were determined by an automatic biochemistry analyzer.

Insulin, leptin, GIP (Gastric inhibitory peptide), amylin, PYY (Peptide YY), and PP (Pancreatic polypeptide) were assayed using a human gut hormone multiplex kit according to the manufacturer's instructions (Millipore, Billerica, MA). Intra-assay variation was lower than 11%. Adiponectin levels were measured by radioimmunoassay (Millipore, Billerica, MA) and the intra-assay variation for this was from 6.90 to 9.25%. Total plasma ghrelin levels were measured using an ELISA assay kit from Millipore according to the manufacturer's instructions. Glucagon levels were assayed using a Millipore RIA kit. Brain-derived neurotrophic factor (BDNF), GH, Agouti-related protein (AgRP), and prolactin were measured with a Human Brain-Derived/Pituitary Protein Multiplex Panel assay kit according to the manufacturer's instructions (Millipore): intra-assay variation was lower than 10%. Cytokines were assayed using a human cytokine/chemokine assay kit according to the manufacturer's instructions (Millipore): intra-assay variation was lower than 10.5%. C-reactive protein (CRP) was measured using an ALPCO ELISA kit according to the manufacturer's instructions (ALPCO Diagnostics, Salem NH), and the intra-assay variation was 5.5–6.0%.

5-Bromo-2-deoxy Uridine (BrdU) has been proven to be a suitable marker for proliferating cells in numerous in vitro studies as well as in vivo studies. In this study, the level of cell proliferation in GC-1 cells at 3 d after RF and/or X-ray treatment were determined by BrdU enzyme linked immunosorbent (ELISA) assay kit (Calbiochem Merck, Whitehouse Station, NJ, USA). Briefly, 100 μL cells at 1 × 10⁵/mL were seeded per well into a 96-well culture dish, and allowed BrdU to label cells for 24 h in the incubator. After that, anti-BrdU antibody was added and incubated for 1 h at room temperature, then the reconstituted peroxidase goat anti-mouse immunoglobulin G (IgG) horseradish peroxidase (HRP) was added to the well. Finally, the absorbance of the reacted product was measured by using a spectrophotometric plate reader at dual wave lengths of 450–540 nm (Bio-Rad).

The levels of IL-1β in the serum and BALF, tumor necrosis factor-α (TNF-α) and IL-10 in the BALF were measured by commercially available ELISA assay kits (Sigma, USA), according to the manufacturer’s instructions.