Approximately 50 mg of heart muscle (ventricle) or soleus muscle were homogenized in QIAZOL (Qiagen, Germany) plus 8 µl proteinase K (Qiagen) in a TissueLyser bead mixer (Qiagen) at 25 Hz in two 5-min repetitions. Total RNA isolation was performed with an EZ1 RNA Universal Tissue Kit and Biorobot EZ1 (Qiagen) in accordance with the manufacturer's instructions. Total RNA concentrations were measured at 260 nm via spectrophotometry (ND-1000 spectrophotometer, NanoDrop Technologies, Inc.). Samples were frozen and stored at −80°C for subsequent analysis.
Tissuelyser bead mixer
Manufactured by Qiagen
Sourced in United States
The TissueLyser bead mixer is a lab equipment used for the homogenization of biological samples. It utilizes high-speed agitation of samples in the presence of beads to effectively disrupt and homogenize various tissue types.
Automatically generated - may contain errors
Lab products found in correlation
Oxytocin eia kit, by Phoenix Pharmaceuticals (2 mentions)
Nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Biorobot ez1, by Qiagen (1 mentions)
Proteinase k, by Qiagen (1 mentions)
Qiazol, by Qiagen (1 mentions)
Ez1 rna universal tissue kit, by Qiagen (1 mentions)
Powerwave xs spectrophotometer, by Agilent Technologies (1 mentions)
Cathepsin k activity fluorometric assay kit, by Abcam (1 mentions)
Ls50b, by PerkinElmer (1 mentions)
5 protocols using tissuelyser bead mixer
1
Cardiac and Skeletal Muscle RNA Extraction
2
Oxytocin Protein Quantification in Heart
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For the measurements of the OT and OTR protein concentration in the heart’s homogenates following EIA Kits were used: Oxytocin EIA Kit (Phoenix Pharmaceuticals Inc., USA), Oxytocin Receptor EIA Kit (Sunred Biological Technology Co. Ltd., China). Each sample of tissue was homogenised in a TissueLyser bead mixer (Qiagen, USA) and centrifuged (10,000 rpm, 10 min, 4°C). The supernatant was collected and frozen at –80°C until the analysis. Total protein concentration was measured using bicinchoninic acid (BCA) Protein Assay Kit (Pierce, Holland), according to the manufacturer’s instructions. Results obtained were presented as an absolute ratio: concentration/total protein concentration (×10–9) for OT, OTR. Plasma OT concentration was measured for plasma samples from each group using an Oxytocin ELISA Kit (Phoenix Pharmaceuticals Inc., USA).
3
Homogenization and Protein Quantification
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Due to the limited amount of sample, homogenization of each sample was performed as follows. All samples were homogenized in water in a TissueLyser bead mixer (Qiagen) and centrifuged twice at 7826 g for 10 min at 4°C. Plasmin activity and elastase activity were assayed directly after centrifugation. Supernatants were stored at −80°C for further analyses of cathepsin K, elastin, and total protein content.
For the determination of elastin levels, samples of heart muscle were homogenized in phosphate-buffered saline in accordance with the manufacturer's (see below) instructions and stored overnight at −20°C. After two freeze-thaw cycles, the homogenates were centrifuged for 5 min at 5000g . The supernatant was removed and assayed immediately as described below.
Total protein concentration was measured at 562 nm on a BioTek Power Wave XS spectrophotometer (BioTek Instruments, USA) using the bicinchoninic acid Protein Assay Reagent (Pierce, Holland) in accordance with the manufacturer's instructions.
For the determination of elastin levels, samples of heart muscle were homogenized in phosphate-buffered saline in accordance with the manufacturer's (see below) instructions and stored overnight at −20°C. After two freeze-thaw cycles, the homogenates were centrifuged for 5 min at 5000
Total protein concentration was measured at 562 nm on a BioTek Power Wave XS spectrophotometer (BioTek Instruments, USA) using the bicinchoninic acid Protein Assay Reagent (Pierce, Holland) in accordance with the manufacturer's instructions.
4
Cardiac Oxytocin and NT-proBNP Quantification
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The following EIA Kits were used for the evaluation of oxytocin, oxytocin receptor concentration in the heart's homogenates, and N-terminal prohormone B-type natriuretic peptide (NT-proBNP) in plasma: Oxytocin EIA Kit (Phoenix Pharmaceuticals Inc., USA), Oxytocin Receptor EIA Kit (Sunred Biological Technology Co. Ltd., China), and NT-proBNP (Wuhan EIAab Science Co. Ltd., China). For the ventricle cardiac muscle, each sample of tissue was homogenized in the TissueLyser bead mixer (Qiagen, USA) and centrifuged (10 000 rpm for 10 minutes, 4°C). The supernatant was collected and frozen at −80°C until analysis. Total protein concentration was measured using bicinchoninic acid (BCA) Protein Assay Kit (Pierce, Holland), according to the manufacturer's instructions. Results obtained were presented as an absolute ratio: concentration/total protein concentration (×10−9) for OT and OTR and total protein concentration (pg/mL) for plasma NT-proBNP.
5
Enzyme Activity Quantification Protocol
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All samples were homogenized in water in a TissueLyser bead mixer (Qiagen) and centrifuged twice at 10,000 rpm for 10 min at 4 °C. Plasmin activity and elastase activity were assayed directly after centrifugation. Supernatants were stored at -80 °C for further analyses of cathepsin K and total protein content.
The total protein concentration was measured at 562 nm on a BioTek Power Wave XS spectrophotometer (BioTek Instruments, USA) using the bicinchoninic acid (BCA) Protein Assay Reagent (Pierce, Holland) in accordance with the manufacturer's instructions.
Assays of enzyme activity. Enzyme activity was measured using a spectrofluorimeter (LS-50B, PerkinElmer, USA). Fluorescence measurements were performed with induction at λ = 355 nm and emission at λ = 460 nm. The substrate for elastase activity was Z-Arg-Arg-7-amido-4-methylcoumarin, and the substrate for plasmin was Boc-Val-Leu-Lys-7-amido-4-methylcoumarin (Bachem, Biochemica GmbH, Germany). A commercial kit (Cathepsin K Activity Fluorometric Assay Kit, BioVision, Inc., USA) was used to measure cathepsin K activity (substrate: Ac-Lys-Arg-amino-4-trifluoromethyl coumarin) with a 400-nm excitation filter and a 505-nm emission filter. The results are presented as the ratio of enzyme fluorescence to total protein concentration.
The total protein concentration was measured at 562 nm on a BioTek Power Wave XS spectrophotometer (BioTek Instruments, USA) using the bicinchoninic acid (BCA) Protein Assay Reagent (Pierce, Holland) in accordance with the manufacturer's instructions.
Assays of enzyme activity. Enzyme activity was measured using a spectrofluorimeter (LS-50B, PerkinElmer, USA). Fluorescence measurements were performed with induction at λ = 355 nm and emission at λ = 460 nm. The substrate for elastase activity was Z-Arg-Arg-7-amido-4-methylcoumarin, and the substrate for plasmin was Boc-Val-Leu-Lys-7-amido-4-methylcoumarin (Bachem, Biochemica GmbH, Germany). A commercial kit (Cathepsin K Activity Fluorometric Assay Kit, BioVision, Inc., USA) was used to measure cathepsin K activity (substrate: Ac-Lys-Arg-amino-4-trifluoromethyl coumarin) with a 400-nm excitation filter and a 505-nm emission filter. The results are presented as the ratio of enzyme fluorescence to total protein concentration.
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