cGMP levels were measured using the cGMP enzyme immunoassay (EIA) kit (#581021, Cayman Chemical). Isolated adult murine cardiomyocytes were homogenized in 100 µl of 0.1 M HCl and centrifuged at 1000 × g for 10 min. The supernatants were diluted 1:6 with diluent buffer and used for cGMP level measurements according to the manufacturer’s protocol. For the measurement of cAMP levels using the cAMP enzyme immunoassay (EIA) kit (#581001, Cayman Chemical) the samples were processed as described above. The plasma Musclin concentration was determined by the mouse Osteocrin ELISA (#EKC37526, Biomatik), the ANP concentrations in plasma by the ANP-Fluorescent EIA kit (Phoenix Pharmaceuticals, #FEK-005-24) and the CNP plasma concentration using the CNP ELISA kit (#MBS2514795, detecting mature CNP peptides, MyBioSource) according to manufacturer’s instructions.
Eia kit
Manufactured by Cayman Chemical
Sourced in United States
The EIA kit is a laboratory tool used to detect and quantify specific molecules in a sample through enzyme-linked immunosorbent assay (ELISA) technique. It provides a standardized and reliable method for analyte measurement.
Automatically generated - may contain errors
Lab products found in correlation
Lipoxygenase inhibitor screening assay kit, by Cayman Chemical (4 mentions)
Vacuum manifold, by Waters Corporation (2 mentions)
Oasis hlb column, by Waters Corporation (2 mentions)
Dnp48 hsa, by Merck Group (2 mentions)
Enzyme immunoassay, by Cayman Chemical (2 mentions)
Arachidonic acid, by Cayman Chemical (2 mentions)
Speed vac, by Cayman Chemical (2 mentions)
Eia buffer, by Cayman Chemical (2 mentions)
Raw 264.7 cell, by Korean Cell Line Bank (2 mentions)
Oxyblot protein oxidation detection kit, by Merck Group (2 mentions)
Multiskanascent analyzer, by Thermo Fisher Scientific (2 mentions)
Vacutainer sampling tubes, by BD (2 mentions)
Gm csf, by R&D Systems (2 mentions)
Standard 20e, by Merck Group (2 mentions)
Multiskan fc, by Thermo Fisher Scientific (2 mentions)
Quantikine hs elisa human il 6 immunoassay, by R&D Systems (1 mentions)
Human p selectin cd62p elisa kit, by R&D Systems (1 mentions)
Quantikine hs elisa human tnf α immunoassay, by R&D Systems (1 mentions)
Cytation 5 imaging plate reader, by Agilent Technologies (1 mentions)
Testosterone eia kit, by Cayman Chemical (1 mentions)
244 protocols using eia kit
1
Measuring Cardiac Signaling Molecules
2
Quantification of LTB4 in Neutrophils
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Total LTB4 levels were measured using an ELISA kit (R&D Systems). Briefly, neutrophils or PLB-985 cells were resuspended at 1 × 106 cells/ml in PBS and incubated for 30 min on ice. GM–CSF (5 ng/ml; R&D Systems) was added, and cells were further incubated for 1 h at 37°C, centrifuged at 400xg for 5 min, resuspended in a volume of 200 μl at 24 × 106 cells/ml in RPMI, and incubated at 37°C until stimulated. The reactions were stopped by adding cold PBS. Cells were centrifuged and LTB4 in supernatants were assayed according to manufacturer’s instructions. Exosomal LTB4 was measured using the EIA kit according to manufacturer’s protocol (Cayman Chemicals) after disruption by sonication. To measure LTB4 in the vesicular fractions of lysed neutrophils, total lipids were first extracted from the different fractions using a method outlined by McColl and colleagues [52 (link)]. The extracted lipids were acidified to pH 4.0, loaded onto a C18 SPE column, and eluted with ethyl acetate containing 10% methanol, evaporated under inert nitrogen stream, and resuspended in buffer to obtain a concentrated pool of LTB4. LTB4 levels were measured using the EIA kit (Cayman Chemicals) according to manufacturer’s recommendations.
3
Quantification of Leukotriene B4 in Neutrophils
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Total LTB4 levels were measured using an ELISA kit (R&D Systems). Briefly, neutrophils or PLB-985 cells were resuspended at 1 x 106 cells/ml in PBS and incubated for 30 min on ice. GM-CSF (5 ng/ml; R&D Systems) was added and cells were further incubated for 1 hr at 37°C, centrifuged at 400 xg for 5 min, resuspended in a volume of 200 μl at 24 x 106 cells/ml in RPMI and incubated at 37°C until stimulated. The reactions were stopped by adding cold PBS. Cells were centrifuged and LTB4 in supernatants were assayed according to manufacturer’s instructions. Exosomal LTB4 was measured using the EIA kit according to manufacturer’s protocol (Cayman Chemicals) after disruption by sonication. To measure LTB4 in the vesicular fractions of lysed neutrophils, total lipids were first extracted from the different fractions using a method outlined by McColl et al. [55 (link)]. The extracted lipids were acidified to pH 4.0 loaded onto a C18 SPE column and eluted with ethyl acetate containing 10% methanol, evaporated under inert nitrogen stream and resuspended in buffer to obtain a concentrated pool of LTB4. LTB4 levels were measured using the EIA kit (Cayman chemicals) according to manufacturer’s recommendations.
4
Quantifying Ovarian PGE2 Levels
5
Biomarker Measurement in Aspirin Therapy
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All blood samples were taken in fasting condition at 9 a.m. at least 2 h after the last dose of ASA. Standard laboratory techniques were used for regular laboratory testing. Serum BDNF concentrations were measured using BDNF Quantikine Immunoassay (R&D Systems, USA) as previously described for our laboratory [14 (link)]. ELISA kits were also used to determine concentrations of the following parameters: serum TXB2 (EIA kits, Cayman Chemicals, Ann Arbor, MI, USA), von Willebrand factor (vWF) molecule (vWF: Ag), tumor necrosis factor (TNF)-α (Quantikine® HS ELISA Human TNF-α Immunoassay), interleukin (IL)-6 (Quantikine® HS ELISA Human IL-6 Immunoassay; both R&D Systems, Inc., Minneapolis, USA), soluble CD40 ligand (sCD40L; Human soluble CD40 Ligand Immunoassay, R&D Systems, Inc., NE, USA), and soluble P-selectin (human P-selectin/CD62P ELISA kit R&D Systems, Inc., Minneapolis, USA). High-sensitivity C-reactive protein (hsCRP) concentrations were assessed using a Cobas Integra 800 device (Roche, Basel, Switzerland), as previously described [21 (link),22 (link)]. The compliance with ASA treatment was defined according to previously described criteria [20 (link)].
6
Quantification of LTB4 and PGE2 in BALF
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The concentrations of LTB4 and PGE2 in the BALF supernatants were assayed using enzyme immunoassay (EIA) kits according to the manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI, USA).
7
Plasma Steroid Hormone Measurement
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Plasma levels of 11‐keto testosterone (11‐KT), maturation inducing hormone (MIH) and testosterone (T) were measured using enzyme immuno‐assay (EIA) kits (Cayman chemical, USA). Steroid sex hormones were extracted from frozen plasma samples using diethyl ether. Briefly, 500‐µL plasma was thoroughly mixed with diethyl ether (1:4 plasma:solvent) by vortexing. The phases were separated by freezing in liquid nitrogen, and the organic phase was decanted. The process was repeated, and the combined organic phase was evaporated overnight at 25°C. The dry extracts containing steroid hormones were resuspended in 500 µL of EIA buffer and frozen at −80°C pending analysis. Samples were analysed in duplicate as described by kit manufacturer. Absorbances were read at 410 nm for all three hormones using a Cytation 5 Imaging plate reader (BioTek Instruments, USA). Plasma steroid levels were calculated from standard curves fitted with linear regression of logit transformed data as instructed by the manufacturer.
8
Serum Deprivation and Vasopressin Stimulation of Cholangiocytes
9
Serum Estrogen and Progesterone Measurement
10
Serum and Intratesticular Hormone Levels
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Blood was collected from 6 month-old male mice by cardiac puncture, left at room temperature for ~15 min., and centrifuged. Serum was stored at −20 °C. Serum testosterone and estradiol from Mras−/− males was measured using EIA kits (Cayman Chemical). Intratesticular testosterone was extracted from whole testes of 6 month-old male mice. Testes were first mechanically lysed using small glass tissue grinders in 500 µL of RIPA buffer (150 mM NaCl, 50 mM Tris–HCl pH 8.0, 1 % NP-40, 0.5 % Na-deoxycholate, 0.1 % SDS) plus protease inhibitors (Roche). Testosterone was extracted from the tissue lysates with three successive extractions with 2.5 mL ether each. The ether extract was dried and pellets resuspended in EIA buffer for analysis by the Testosterone EIA kit (Cayman). We determined serum testosterone levels both in male mice that had undergone the resident-intruder test on the same day, and in mice that had not. There were no significant differences in testosterone levels between these mice. Serum estradiol was measured in males that had not undergone behavioral testing. Serum testosterone, n = 26, WT; n = 31, Mras−/−. Total testosterone per mg testis tissue, n = 14, WT; n = 19, Mras−/−. Serum testosterone in aggressive and non-aggressive Mras−/− males, n = 9 and n = 10, respectively. Serum estradiol, n = 12 each.
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