B0560

Manufactured by Merck Group

The B0560 is a laboratory equipment product offered by Merck Group. It is designed for general laboratory use. The core function of this product is to provide a precise and reliable method for [core function]. Further details about the intended use or specific applications of this product are not available.

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Blebbistatin (B0560), (6 citations)

5 protocols using b0560

Preparation and Characterization of Compounds

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Omecamtiv mecarbil and (−)‐blebbistatin were purchased from Selleck (S2623) and Sigma (B0560), respectively. Stock solutions were prepared in DMSO (molecular biology grade, Sigma, D8418) and compound purities were estimated to be >95% by RP‐HPLC and ESI mass spectrometry.

Kampourakis T., Zhang X., Sun Y, & Irving M. (2017). Omecamtiv mercabil and blebbistatin modulate cardiac contractility by perturbing the regulatory state of the myosin filament. The Journal of Physiology, 596(1), 31-46.

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Actin and Myosin Inhibition for Merozoite Invasion

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Where indicated, erythrocytes were pre-treated with the actin and myosin inhibitors cytochalasin D (C2618, Sigma), blebbistatin (B0560, Sigma) and 2,3-Butanedione monoxime (BDM, B0753, Sigma) prior to merozoite invasion for 1 h at 37 °C, and washed two times. Filtered merozoites were then added to treated erythrocytes and invasion assays completed as published32 (link).
To deplete erythrocytes of ATP prior to invasion, erythrocytes were washed three times in PBS and incubated at 8% haematocrit in either PBS alone or depletion media containing 6 mM iodoacetamide (I6125, Sigma) and 10 mM inosine (I4125, Sigma) in PBS for between 10 min and 3 h at 37 °C in a humidifying oven on rotation. Treated cells were then washed three times in incomplete culture medium and used in merozoite invasion assays.

Zuccala E.S., Satchwell T.J., Angrisano F., Tan Y.H., Wilson M.C., Heesom K.J, & Baum J. (2016). Quantitative phospho-proteomics reveals the Plasmodium merozoite triggers pre-invasion host kinase modification of the red cell cytoskeleton. Scientific Reports, 6, 19766.

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Establishing and Treating Cell Lines

Cited 1 time

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Human HT-1080 fibrosarcoma, A431 epidermoid carcinoma, MCF-7 breast cancer, and human embryonic kidney (HEK) 293T cells were obtained from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing glucose (4.5 g/liter), l-glutamine, and sodium pyruvate (11995073, Gibco). Medium was supplemented with 10% heat-inactivated FBS (16140071, Gibco) and 1% penicillin-streptomycin (10,000 U/ml; 15140122, Gibco). Cells were maintained at 37°C with 5% CO₂. A431 cells expressing GFP-E-cad were a gift from V. Bruntons (EH4 2XR, University of Edinburgh, UK) (63 (link)). In select experiments, cells were treated with the following pharmacological agents: Y-27632 (20 μM; Y0503, Sigma-Aldrich), ionomycin (0.5 and 1 μM; I0634, Sigma-Aldrich), blebbistatin (50 μM; B0560, Sigma-Aldrich), Rho inhibitor I (1 μg/ml; CT04-A, Cytoskeleton Inc.), LPA (50 μM; L7260, Sigma-Aldrich), paclitaxel (taxol equivalent) (1 μM; P3456, Thermo Fisher Scientific), colchicine (125 μM; C9754, Sigma-Aldrich), and importazole (50 μM; SML0341, Sigma-Aldrich).

Law R.A., Kiepas A., Desta H.E., Perez Ipiña E., Parlani M., Lee S.J., Yankaskas C.L., Zhao R., Mistriotis P., Wang N., Gu Z., Kalab P., Friedl P., Camley B.A, & Konstantopoulos K. (2023). Cytokinesis machinery promotes cell dissociation from collectively migrating strands in confinement. Science Advances, 9(2), eabq6480.

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Murine Cardiac Optical Mapping

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Mice were anesthetized with isoflurane to the surgical plane of anesthesia, and heparin was injected intraperitoneally (100 units) before cervical dislocation. The heart was then removed and washed in cold, oxygenated (95% O₂, 5% CO₂) Tyrode’s solution (137mM NaCl; 5.4mM KCl; 1mM MgCl2; 5 mM HEPES; 10 mM glucose; 3 mM NaOH; 2.5mM Ca²⁺; pH 7.4). The aorta was cannulated with a 21-gauge cannula, and the heart was retrogradely perfused with Tyrode’s solution, maintaining aortic pressure between 80 and 120 mmHg. An electrode (Harvard Apparatus) was placed on the surface of the right atrium for pacing stimulations (10 Hz, 12 Hz, and 14 Hz) generated by PowerLab 26T (ADInstruments). To eliminate contractile artifacts, hearts were loaded with blebbistatin (Sigma-Aldrich, B0560-5 mg, 50 μl of 2.5 mg/ml in dimethyl sulfoxide [DMSO]). We then perfused the hearts with the voltage-sensitive dye di-4-ANEPS (Invitrogen, D-1199, 20 μl of 2.5 mg/ml in DMSO). An LED light was used for excitation (wavelength, 530 nm). Fluorescence emission, signifying Vm, was long-pass filtered (>590nm, 590FG05-50, Andover Corporation, Optical Filter) and measured with a MiCAM0 CMOS camera (SciMedia). Surface electrocardiograms (ECGs) (ADInstruments) were monitored during experiments by using LabChart. Conduction velocities and activation maps were calculated with Rhythm software(Laughner et al., 2012 (link)) .

Monroe T.O., Hill M.C., Morikawa Y., Leach J.P., Heallen T., Cao S., Krijger P.H., de Laat W., Wehrens X.H., Rodney G.G, & Martin J.F. (2019). YAP Partially Reprograms Chromatin Accessibility to Directly Induce Adult Cardiogenesis in Vivo. Developmental cell, 48(6), 765-779.e7.

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Spheroid Formation and Cytoskeleton Modulation

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To generate MCSs, cells were trypsinized using 0.25 g per l trypsin (Sigma-Aldrich, T4049) and 2000 cells were transferred to a low adhesion U-bottom 96-well plate (BRAND, 781900) per well with 250 μl medium. They were incubated for about 24 hours during which the spheroids formed.⁴ To alter the cytoskeleton of cells in spheroids, 25 μM (−)-blebbistatin (Sigma-Aldrich, B0560) or 100 nM cytochalasin D (Sigma-Aldrich, C8273) was added to the respective culture medium for 24 hours during spheroid formation, avoiding the drug concentration that cells are exposed to varies from the spheroid surface to the core. As spheroid formation and drug treatment are time dependent (see Fig. S1, ESI†), the time period to start observation is set for 24 hours in this study to guarantee the comparability throughout the used model systems.

Xie X., Sauer F., Grosser S., Lippoldt J., Warmt E., Das A., Bi D., Fuhs T, & Käs J.A. (2024). Effect of non-linear strain stiffening in eDAH and unjamming. Soft Matter, 20(9), 1996-2007.

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