Circulating RLX was analyzed in EDTA-containing serum of RLX-infused rats using Human Relaxin-2 Quantikine® ELISA Kit (R & D Systems, Minneapolis, MN) according to the manufacturer’s instructions. The assay is specific for human relaxin-2, which means, it does not significantly cross-react with rat RLX. The sensitivity of the assay is ~ 7.81 pg/ml (equivalent to the lowest standard), the maximum dose for accurate determination was 500 pg/ml as reported by R & D Systems. Therefore, serum samples were diluted 1:10–1:50 with phosphate-buffered saline (PBS), so that values would fall on the standard curve. Optical density was measured at 570 nm using a SpectraCount™ (Packard Instrument Company Inc., Meriden, CT). Serum concentrations [ng/ml] were obtained after correction of the readings by the zero standard and multiplication with the dilution factor.
Human relaxin 2 quantikine elisa kit
Manufactured by R&D Systems
Sourced in United Kingdom, United States
The Human Relaxin-2 Quantikine ELISA Kit is a quantitative sandwich enzyme immunoassay designed to measure human relaxin-2 levels in cell culture supernates, serum, and plasma.
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4 protocols using human relaxin 2 quantikine elisa kit
1
Quantification of Circulating Relaxin-2
2
Biomarker Analysis in Peripartum Cardiomyopathy
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Blood samples were collected in S-Monovette® tubes containing ethylenediaminetetraacetic acid or clot activator at the time point of first diagnosis in PPCM patients and from pregnancy stage-matched healthy controls (pre- and post-partum, time points of blood sampling as indicated above) and age matched non-pregnant women. Laboratory workup was performed as routine investigation by hospital laboratories for N-terminal pro-brain natriuretic peptide (NT-proBNP). For analyses of relaxin levels, serum was separated by centrifugation at 500 × g for 10 min and aliquots were stored at −80 °C. Relaxin-2 was measured using the Human Relaxin-2 Quantikine ELISA Kit (R&D Systems) strictly according to the manufacturer’s protocol. Mouse serum Relaxin-1 levels were quantified using the mouse Relaxin-1 ELISA Kit purchased from ORIGENE following the provided protocol. Mouse Prolactin was quantified from plasma samples using the mouse Prolactin ELISA Kit from Abcam according to the manufacturer’s protocol.
3
Cytokine and Hormone Analysis in Renal Cell Carcinoma
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We analyzed the presence of two cytokines (IL-6 and IL-8) and testosterone in the serum of 25 ccRCC and 9 pRCC patients using Solid-Phase Chemiluminescent Immunometric Assay kits with an Immulite 1000 Immunoassay System and ADVIA Centaur CP (all Siemens Healthcare GmbH, Erlangen, Germany) according to manufacturer’s instructions.
Serum levels of RLN2 were analyzed with a Human Relaxin-2 Quantikine ELISA Kit (R&D Systems, Abingdon, United Kingdom) according to manufacturer’s instructions. Assay Diluent was mixed with the standard, control or samples in a provided 96-well plate and incubated for 2 hrs at RT. After washing, the Human Relaxin-2 Conjugate was added to each well and incubated for 2 hrs at RT. Following repeated washing and 30 min incubation with Substrate Solution, the reaction was stopped with Stop Solution and the optical density was determined using an Infinite M Plex microplate reader (Tecan Deutschland GmbH, Crailsheim, Germany).
Serum levels of RLN2 were analyzed with a Human Relaxin-2 Quantikine ELISA Kit (R&D Systems, Abingdon, United Kingdom) according to manufacturer’s instructions. Assay Diluent was mixed with the standard, control or samples in a provided 96-well plate and incubated for 2 hrs at RT. After washing, the Human Relaxin-2 Conjugate was added to each well and incubated for 2 hrs at RT. Following repeated washing and 30 min incubation with Substrate Solution, the reaction was stopped with Stop Solution and the optical density was determined using an Infinite M Plex microplate reader (Tecan Deutschland GmbH, Crailsheim, Germany).
4
Serelaxin Effects on Murine Metabolic Parameters
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After two weeks of treatment with either placebo or serelaxin, mice were anaesthetised by a cocktail of ketamine (85 mg/kg) and xylazine (8.5 mg/kg) via intraperitoneal injections followed by cardiac puncture for blood collection. Two μL of whole blood was used to measure HbA1c using a cobas b 101 POC system (Roche, Basel, Switzerland). Blood plasma was used to determine plasma osmolality using a vapour pressure osmometer (VAPRO Model 5600, Wescor Inc, Logan, Utah) and concentrations of serelaxin using a Human Relaxin-2 Quantikine ELISA kit (Cat# DRL200; R&D systems, Minneapolis, MN, USA) following manufacturer’s protocol with detection sensitivity of 4.57 pg/mL. Mesenteric arcade, whole heart and aorta were isolated and immediately placed in ice cold Krebs bicarbonate solution (120 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, 11.1 mM D-glucose, 2.5 mM CaCl2) and then cleared of fat and connective tissues.
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