Plasma brain natriuretic peptide (BNP) and Ang II were determined to assess myocardial stretch and renin–angiotensin–aldosterone system (RAAS) system activity, respectively, using available enzyme-linked immunosorbent assay (ELISA) kits (Phoenix Pharmaceuticals, Burlingame, CA, USA). The levels of plasma epinephrine (E) and norepinephrine (NE) were measured to analyze the sympathetic response with commercially available ELISA kits (KA3746, Abnova, Taipei, Taiwan). NE concentrations in the LA, LV, aorta and kidney tissues were also determined (KA3836, Abnova, Taipei, Taiwan). The levels of IL-6, TNF-α and cGMP in the aorta were assessed via available ELISA kits (Abcam, ab-100573, ab-100756 and ab-65356, respectively). Plasma total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), HDL and glycosylated hemoglobin (HbA1c) were measured with specific kits according to the manufacturer’s instructions (Roche Diagnostics, Madison, WI, USA). Urinary electrolyte and osmolality levels were measured by the autoanalyzer CX5 (Beckman Instruments, Fullerton, CA, USA). Urinary glucose and uric acid levels were measured by Cobas Mira autoanalyzer (Roche Diagnostics, Madison, WI, USA).
Ab65356
Manufactured by Abcam
Sourced in United States
Ab65356 is a laboratory equipment product. It is designed for a specific core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation. Therefore, a detailed description is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ka3836, by Abnova (1 mentions)
Ab 100573, by Abcam (1 mentions)
Ab 100756, by Abcam (1 mentions)
Cobas mira autoanalyzer, by Roche (1 mentions)
Halt protease phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
S nitroso n acetylpenicillamine snap, by Merck Group (1 mentions)
5 protocols using ab65356
1
Biomarkers of Myocardial Stress and Inflammation
2
Murine Tissue Harvesting and Serum Analysis
3
Cardiomyocyte-Endothelial Co-culture Assay
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Adult primary cultured cardiomyocytes were isolated from mouse hearts following the protocol described by Ackers-Johnson et al [40 (link)] and C166 mouse yolk sac endothelial cells were from American Type Culture Collection (ATCC) (Manassas, VA, USA). Cells were incubated in the presence or absence of linagliptin (9 nmol/L) for 5 min for immunoblotting experiments and for 24 h for RNA sequencing. In addition, cardiomyocytes were incubated in the presence or absence of 100 µmol/L S-nitroso-N-acetylpenicillamine (SNAP) (Sigma-Aldrich, Oakville, Ontario, Canada) [41 (link)] for 5 min prior to immunoblotting. RNA isolation from cardiomyocytes was performed using TRIzol Reagent (Life Technologies, Carlsbad, CA, USA). For co-culture experiments, adult primary cultured cardiomyocytes and C166 cells were cultured separately on opposite sides of Millicell 6-well plate inserts with a 10 µm pore size PET membrane (MCRP06H48, EMD Millipore, Billerica, MA, USA) at a 1:2 density under control conditions or with culture media supplemented with 9 nmol/L linagliptin for 5 min. Cardiomyocyte cGMP concentrations were determined by direct immunoassay (ab65356, Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions.
4
Measuring PDE5 Activity in Tissue
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The CC tissue was immediately frozen in liquid nitrogen and crushed into a powder with grinding rods. Then, the powder was placed into a centrifuge tube containing lysis buffer and vortexed for 30 seconds. After storage at 4°C for 30 minutes, the homogenate was centrifuged for 15 minutes at 12 000 g; then, the supernatant was used for protein quantification using the BCA method and lysates containing 5 mg of total protein were used for the following PDE5 activity assay.
Buffer containing Tris (50 mmol/L, pH 7.5), indomethacin (10−5 mol/L), nitro‐l ‐arginine (10−4 mol/L), MgCl2 (10−5 mol/L), bovine serum albumin (0.3 mg/mL) and cGMP (guanosine 3′, 5′‐cyclic monophosphate sodium salt, 2 × 10−6 mol/L) were added into tissue lysates and then incubated at 30°C for 15 minutes. After stopping the reaction via administration of HCl (250 mmol/L), the level of excess cGMP in the reaction solution was determined by specific commercial ELISA kit (ab65356; Abcam). As cGMP was hydrolysed by PDE5, the amount of cGMP detected was negatively correlated with the activity of PDE5.
Buffer containing Tris (50 mmol/L, pH 7.5), indomethacin (10−5 mol/L), nitro‐
5
cGMP Quantification in Cell Lysates
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The cGMP levels in the cell lysates were assayed according to the manufacturer’s protocol (Abcam; ab65356). Six replicates were made for each group. The concentration of cGMP in the samples was then determined by comparing the OD value of the samples to the standard curve.
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