Blood samples were collected into 1.5 Eppendorf tubes or BD Vacutainer® SST™ serum separation tubes (BD, Franklin Lakes, New Jersey), by cardiac puncture from adults (p=70 ±5 days) ephrin-A5−/− (n=13), and wild-type (n=15) male mice and allowed to clot. The blood was then centrifuged to isolate the serum, which was then extracted using diethyl ether (Sigma, St. Louis, MO). Testosterone concentrations were measured using a commercially available competitive ELISA kit (Cayman Chemical Ann Arbor, MI, Cat No.582701).
Competitive elisa kit
Manufactured by Cayman Chemical
Sourced in United States
The Competitive ELISA kit is a laboratory tool used for the quantitative measurement of analytes in a sample. It utilizes the principle of competitive binding to determine the concentration of the target analyte. The kit provides the necessary reagents and protocols to perform the assay.
Automatically generated - may contain errors
Lab products found in correlation
Vacutainer sst serum separation tubes, by BD (1 mentions)
Diethyl ether, by Merck Group (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Microplate reader, by Thermo Fisher Scientific (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Duoset elisa, by R&D Systems (1 mentions)
Ria kit, by MP Biomedicals (1 mentions)
Gamma counter, by PerkinElmer (1 mentions)
9 protocols using competitive elisa kit
1
Testosterone Quantification in Ephrin-A5 Knockout Mice
2
Resolvin and Lipoxin Quantification in Lesions
3
Measuring Estradiol and Progesterone in Granulosa Cells
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After the treatment with DLPC for 24 h, the cultured medium of granulosa cells was collected to measure 17 beta-estradiol and progesterone using competitive ELISA kit (Cat#: 582251, Cat#: 582601; Cayman, Ann Arbor, MI, USA). The ELISA procedure was performed according to Cayman’s ELISA kit instructions, and the optical density (OD) values at 420 nM were measured by a microplate reader (Thermo, Waltham, MA, USA). An eight-point standard curve was obtained for each series of analyses. Finally, the concentrations of 17 beta-estradiol and progesterone were calculated using the formula derived from the standard curve in cultured medium.
4
Oxidative Stress Biomarkers in Saliva
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Oxidative stress status was assessed by measuring the total antioxidant capacity (TAOC) and biomarkers of oxidative stress 8-hydroxy-2′-deoxyguanosine (8-OHdG) and malondialdehyde (MDA) in saliva and the activity of some of the main antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD).
GPx and SOD activities and TAOC levels were determined using a competitive ELISA kit (Cayman Chemical Company; Item numbers 703102, 706002, and 709001, resp.) according to the manufacturer's instructions. MDA levels were measured with NWLSS Malondialdehyde Assay (Northwest Life Science Specialities; Catalog number NWK-MDA01) following the manufacturer's instructions. 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels were measured with NWLSS High Sensitivity 8-OHdG ELISA (Northwest Life Science Specialities; Catalog number NWK-MDA01) following manufacturer's instructions.
GPx and SOD activities and TAOC levels were determined using a competitive ELISA kit (Cayman Chemical Company; Item numbers 703102, 706002, and 709001, resp.) according to the manufacturer's instructions. MDA levels were measured with NWLSS Malondialdehyde Assay (Northwest Life Science Specialities; Catalog number NWK-MDA01) following the manufacturer's instructions. 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels were measured with NWLSS High Sensitivity 8-OHdG ELISA (Northwest Life Science Specialities; Catalog number NWK-MDA01) following manufacturer's instructions.
5
Urinary Isoprostane Determination
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Isoprostane (8-iso prostaglandin F2α) levels in urine were determined by the commercial competitive ELISA kit (Cayman Chemical, Ann Arbor, MI, USA) following manufacturer’s instructions. Results are expressed in ng/mL/mmol of creatinine. Sensitivity of the assay was 3 pg/mL.
6
Quantifying Retinal 2'3'-cGAMP Levels
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Cellular levels of 2′3′-cGAMP were measured using a competitive ELISA kit (Cayman, Ann Arbor, Michigan), following manufacture’s guide. Briefly, retinal cells were lysed in M-PER™ mammalian protein extraction reagent (ThermoFisher) and then centrifuged at 12,000 x g for 10 min. The supernatant (cytosolic fraction) was used for 2′3′-cGAMP measurement. Levels of 2′3′-cGAMP were normalized by the total protein concentration in the supernatant as measured by bicinchoninic acid (BCA) assay.
7
Cytokine and Lipid Mediator Profiling
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Concentrations of IL-6, IL-10, IL-12, IFN-γ, and TNF-α were detected using Human IL-6, IL-10, and IL-12 TNF-α ELISA Sets (BD OptEIA™, BD, San Diego, CA, USA) and IFN-γ by DuoSet ELISA (R&D Systems, Minneapolis, MN, USA). PGE2 and LTB4 levels were measured using a competitive ELISA kit from Cayman Chemical Company (Ann Arbor, MI, USA). All assay types were conducted according to the manufacturer's protocol.
8
VEGF-induced cGMP levels in HUVECs
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HUVEC monolayers were preincubated with the indicated TKI for 30 min in Hepes/HSA containing 0.5 mM isobutylmethylxanthine (IBMX). They were then stimulated with VEGF (50 ng/ml) for 20 min in the same medium or left unstimulated. Cells were lysed in 0.1 M HCl and intracellular cGMP levels were determined using the competitive ELISA kit (581,021, Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's instructions.
9
Evaluating Plasma Hormone Profiles
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Plasma progesterone, thyroid (free thyroxine (T4) and triiodothyronine (T3)) and cortisol concentrations were measured using radioimmunoassay (RIA) kits as per the manufacturer’s instructions (MP Biomedicals, Orangeburg, USA), with specific radioactivity measured with a gamma counter (PerkinElmer Life Science, Massachusetts, USA). The average of intra-assay CVs for progesterone, cortisol and free T4 and T3 were 1.7%, 3.5%, 4.7% and 3.7%, respectively. Total insulin-like growth factor 1 (IGF-1) concentrations were quantified using a commercial ELISA kit (R&D systems Inc., Minneapolis, USA) following the manufacturer’s instructions. The intra-assay CV and minimum detectable dose were 9.3% and 0.026 ng/mL, respectively. Plasma growth hormone (GH) concentrations were measured using a commercial pig ELISA kit (USCN, China) according to the manufacturer’s protocols. The sensitivity of this assay was 0.117 ng/mL and the intra-assay CV was 3.9%. Placental tissue progesterone concentrations were measured using a competitive ELISA kit (Cayman Chemical Co., Ann Arbor, MI, USA) according to the manufacturer’s instructions. The intra-assay CV and assay sensitivity were 5.2% and 10 pg/mL, respectively.
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