High-resolution respirometry (Oxygraph-2K; Oroboros Instruments) was used to measure oxygen consumption in permeabilized muscle fibers from the TA muscle (control and DEN) of mice. Briefly, fibers were mechanically separated in ice-cold biopsy preservation solution buffer (2.77 mM CaK2EGTA, 7.23 mM K2EGTA, 7.55 mM Na2ATP, 6.56 mM MgCl2⋅6H2O, 20 mM taurine, 15 mM Na2 phosphocreatine, 20 mM imidazole, 0.5 mM DTT, 50 mM 2-(N-morpholino)ethanesulfonic acid hydrate, and pH 7.1), permeabilized in biopsy preservation solution with 40 μg/μl saponin at 4 °C for 30 min, and washed in buffer Z (105 mM K-2-(N-morpholino)ethanesulfonic acid, 30 mM KCl, 10 mM KH2PO4, 5 mM MgCl2⋅6H2O, 1 mM EGTA, 5 mg/ml bovine serum albumin, and pH 7.4). Fibers were then incubated in the chamber with oxygenated buffer Z supplemented with 10 μM of Amplex-Red to measure ROS production as well as 1 μM blebbistatin (catalog no.: B592500; Toronto Research Chemicals) to prevent tetanus of the muscle and 25 U/ml Cu/Zn SOD1 to convert O2− to H2O2 and 2 mM EGTA. After obtaining background values, substrates were titrated as follows to assess respiration and ROS production: pyruvate–malate (complex I, state 2), ADP (complex I, state 3), and succinate (complex I and II, state 3).
Blebbistatin
Manufactured by LGC
Sourced in Canada
Blebbistatin is a small-molecule inhibitor that selectively binds to and inhibits the myosin II ATPase activity. It is commonly used as a research tool to study the role of myosin II in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Y 27632, by Merck Group (3 mentions)
Oxygraph 2k, by Oroboros (2 mentions)
Axopatch 200b amplifier, by Molecular Devices (2 mentions)
Clampfit 10 data analysis module of the pclamp 10 electrophysiology data acquisition analysis software, by Molecular Devices (2 mentions)
Co2 independent medium, by Thermo Fisher Scientific (2 mentions)
L 1l glutamine, by Quality Biological (2 mentions)
Calyculin a, by Cell Signaling Technology (2 mentions)
A1r microscope, by Nikon (1 mentions)
Rhod 2 am, by Biotium (1 mentions)
Di 4 anepps, by Biotium (1 mentions)
Mcdb 131 medium, by Thermo Fisher Scientific (1 mentions)
Egm 2 bulletkit, by Lonza (1 mentions)
X tremegene hp dna transfection reagent, by Roche (1 mentions)
Ebm 2 culture medium, by Lonza (1 mentions)
Doxycycline, by Merck Group (1 mentions)
4 hydroxyacetophenone, by Merck Group (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 680 phalloidin, by Thermo Fisher Scientific (1 mentions)
Phospho myosin light chain 2 thr18 ser19, by Cell Signaling Technology (1 mentions)
Dylight 488, by Thermo Fisher Scientific (1 mentions)
30 protocols using blebbistatin
1
Muscle Fiber Respiration Profiling
2
Isolation of Murine Cardiomyocytes
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The mouse was anesthetized with 2% inhaled isoflurane. The heart was removed and promptly perfused with a collagenase solution, and cardiomyocytes were isolated as previously described.10 , 14 , 20 2,3‐Butanedione monoxime was replaced by (−)‐blebbistatin (25 μmol/L; Toronto Research Chemicals) in stopping buffer to inhibit cardiomyocyte contraction after isolation.
3
Calcium Handling in RyR2-E4872Q Mutant Mice
4
Optical Mapping of Atrial Conduction
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The heart was excised, and then the coronary artery was perfused ex-vivo with a Krebs solution bubbled with 95% O2, 5% CO2 at 10 mL/min flow rates and 37 °C on a Langendorff apparatus. After a 30-min stabilization and electrical–mechanical decoupling by perfusion of a Blebbistatin (Toronto Research Chemicals Inc., Ontario, Canada) solution of concentration 15 µM, the heart was loaded with a voltage-sensitive dye di-4-ANEPPS (Biotium, CA, USA, 100–150 µL, concentration 2 mg/mL of DMSO). Optical recordings were made in the right atrial region. A CCD (charge-coupled device) camera system (CardioCCD, Redshirt Imaging) recorded the potential-dependent fluorescence image at 2 k Hz [7 (link)]. A pair of bipolar electrodes was used for pacing in the right atrium close to the superior vena cava, and another pair of electrodes was used for a ventricular electrical function monitoring on the ventricles. To estimate conduction properties of the atrial preparation, optical maps were recorded during a 1.5 × threshold current of an atrial stimulation for 2 ms, at basic cycle lengths (BCLs) of 250, 200, 150, 130, 120, 110, 100, 90, 80, 70, 60, and 50 ms.
5
Endothelial Cell Culture and Transfection
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Human dermal microvascular endothelial cells (HMECs; Ades et al., 1992 (link)) were grown in MCDB 131 medium (Gibco) supplemented with 10% FBS, 0.003 mg/ml human EGF, 0.001 mg/ml hydrocortisone, and l -glutamine. Primary human pulmonary arterial endothelial cells were grown in EBM-2 culture medium (Lonza) supplemented with 15% FBS and EGM-2 bullet kit (Lonza) and were used at passages 2–6. All cell lines were maintained at 37°C and 5% CO2.
Endothelial cells were plated on glass-bottom coverslips coated with 0.2% gelatin and transfected at 70–80% confluency using X-tremeGENE HP DNA transfection reagent according to manufacturer’s protocol (Roche). For adenoviral infection, endothelial cells were exposed to the adenoviral particles overnight and were used for live-cell imaging at 24–72 h after infection. The procedure was handled according to National Institutes of Health safety guidelines for materials containing BSL-2 organisms. Cells were treated with 20 µM cRO or 10 µM blebbistatin (Toronto Research Chemicals) for 10 min before experiments.
Endothelial cells were plated on glass-bottom coverslips coated with 0.2% gelatin and transfected at 70–80% confluency using X-tremeGENE HP DNA transfection reagent according to manufacturer’s protocol (Roche). For adenoviral infection, endothelial cells were exposed to the adenoviral particles overnight and were used for live-cell imaging at 24–72 h after infection. The procedure was handled according to National Institutes of Health safety guidelines for materials containing BSL-2 organisms. Cells were treated with 20 µM cRO or 10 µM blebbistatin (Toronto Research Chemicals) for 10 min before experiments.
6
Epithelial Tension Regulation by Myosin II
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To test the role of myosin II in the epithelial tension of polarized epithelium, we treated monolayers of MDCK II Tet-off cells and ZO-1/ZO-2 dKD cells for 15–20 h with 100 µM of the myosin II activity inhibitor blebbistatin (Toronto Research Chemicals, North York, Ontario, Canada), 30 µM of the ROCK inhibitor Y-27632 (Sigma Aldrich), or 2 µM of the MLCK activity inhibitor ML-7 (Sigma Aldrich). To block the expression of full-length ZO-1 rescue on ZO1R cells we treated monolayers with 50 ng ml−1 doxycycline (Sigma Aldrich) and incubate for 2–3 days. To induce full-length ZO-1 rescue on ZO1R cells we washed out multiple times monolayers previously treated with doxycycline and incubate for additional 1–2 days.
7
Modulating Apicobasal Polarity in W4 Cells
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For fix and stain experiments, W4 cells were split onto glass coverslips and incubated for 12 h in the presence of 1 μg/ml doxycycline to induce apicobasal polarity and brush border formation. Induced W4 cells were then incubated with either 20 µM blebbistatin (B592500; Toronto Research Chemicals), 4 nM calyculin A (PHZ1044; Invitrogen), or 1 mM 4-hydroxyacetophenone (278564; Sigma-Aldrich) for 10, 30, or 60 min. After drug incubation, cells were fixed using methods described below. For live-cell imaging experiments, W4 cells were transfected with the appropriate construct and then split onto 35 mm glass bottom dishes (Invitro Scientific; product number, D35-20-1.5-N). Cells were imaged 24–72 h after transfection. Once on the microscope, blebbistatin, calyculin A, or 4-hydroxyacteophenone was added after ∼3–5 min of baseline imaging; cells were then imaged for an additional 20–60 min, depending on the drug treatment.
8
Blebbistatin Sourcing Protocol
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Blebbistatin was obtained from Toronto Research Chemical (TRC).
9
Immunostaining of Myosin Regulatory Proteins
10
Macrophage Differentiation and Cytoskeletal Analysis
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The murine RAW 264.7 macrophage cell line was obtained from the American Type Culture Collection (Manassas, VA) and maintained at 37°C supplied with 5% CO2, in DMEM supplemented with 10% heat-inactivated FBS. For MGC differentiation, AMEM was used instead of DMEM.
LPS, IFNγ, taxol and cytochalasin D were purchased from Sigma-Aldrich Inc. (Oakville, ON). Mouse IL-4 was purchased from PeproTech (Dollard des Ormeaux, QC). Wiscostatin was purchased from Enzo Life Sciences (Brockville, ON). Blebbistatin was purchased from Toronto Research Chemicals (Toronto, ON). M-CSF and RANKL (for BMDM analysis) were obtained from R&D Systems (Minneapolis, MN). For western blotting, rabbit anti-iNOS and rabbit anti-GAPDH-HRP antibodies were purchased from Cell Signaling Technology (Whitby, ON). Rabbit anti-MMP-9 and rabbit anti-cathepsin K antibodies were purchased from Abcam (Toronto, ON). For immunostaining, Alexa-Fluor phalloidin (Invitrogen, Burlington, ON) was used to stain the actin cytoskeleton while rabbit and mouse α-tubulin (Sigma, Oakville, ON) and mouse acetylated α-tubulin (Sigma, Oakville, ON) were used to stain the microtubule network. Mouse GM130 antibody was purchased from BD Transduction Laboratories (San Jose, CA).
LPS, IFNγ, taxol and cytochalasin D were purchased from Sigma-Aldrich Inc. (Oakville, ON). Mouse IL-4 was purchased from PeproTech (Dollard des Ormeaux, QC). Wiscostatin was purchased from Enzo Life Sciences (Brockville, ON). Blebbistatin was purchased from Toronto Research Chemicals (Toronto, ON). M-CSF and RANKL (for BMDM analysis) were obtained from R&D Systems (Minneapolis, MN). For western blotting, rabbit anti-iNOS and rabbit anti-GAPDH-HRP antibodies were purchased from Cell Signaling Technology (Whitby, ON). Rabbit anti-MMP-9 and rabbit anti-cathepsin K antibodies were purchased from Abcam (Toronto, ON). For immunostaining, Alexa-Fluor phalloidin (Invitrogen, Burlington, ON) was used to stain the actin cytoskeleton while rabbit and mouse α-tubulin (Sigma, Oakville, ON) and mouse acetylated α-tubulin (Sigma, Oakville, ON) were used to stain the microtubule network. Mouse GM130 antibody was purchased from BD Transduction Laboratories (San Jose, CA).
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