Actin depolymerising drug latrunculin B (Sigma) and MLCK inhibitor ML-7 (Sigma) were diluted to 0.1 µM and 10 µM, respectively. Cells were incubated with medium containing the drugs for 1 h before fixation. The transfection complex was prepared in Opti-MEM I reduced serum medium (Gibco). The amounts of transfection agent, FuGENE® HD (Roche) and DNA were determined following the manufacturer’s guidelines.
Mlck inhibitor ml 7
Manufactured by Merck Group
Sourced in Canada
MLCK inhibitor ML-7 is a chemical compound used in research laboratories. It functions as an inhibitor of myosin light chain kinase (MLCK), an enzyme involved in the regulation of cellular contractility.
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2 protocols using mlck inhibitor ml 7
1
Actin Depolymerization and MLCK Inhibition
2
Pharmacological Modulation of Airway Contractility
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Precision-cut lung slices were prepared, as previously described [22 ] from BALB/c mice following the HDM sensitization or GSK treatment protocol. After 24-72h in culture, viable slices were used for experimentation. Baseline images were recorded before applying 20 mg/ml MCh to the airway lumen. Images were acquired at 100 frames/sec for 60 seconds using the NIS-Elements B3.0 imaging software (Laboratory Imaging, Czech Republic), Nikon ECLIPSE Ti-S Inverted Microscope (20x objective) and a DIGITAL-SIGHT DS-Ri1 camera (Nikon, Canada), and were stored in .tif format. Airway luminal area was measured using ImageJ (NIH, Bethesda MD) and expressed as a percent of the baseline area.
For the pharmacological inhibitor studies, lung slices were incubated with the following: PI3 kinase inhibitor (Ly294002, 50 μM, Cedarlane, ON), MLCK inhibitor (ML7, 30 μM, Sigma, Canada), Rho Kinase inhibitor (Y2763, 1 μM, Cedarlane, ON) or DMSO diluent for 30 min prior to instillation of MCh. At least 2 lung slices from each mouse were evaluated. Inhibitor and DMSO treatments were performed from serial slices obtained from the same mouse.
For the pharmacological inhibitor studies, lung slices were incubated with the following: PI3 kinase inhibitor (Ly294002, 50 μM, Cedarlane, ON), MLCK inhibitor (ML7, 30 μM, Sigma, Canada), Rho Kinase inhibitor (Y2763, 1 μM, Cedarlane, ON) or DMSO diluent for 30 min prior to instillation of MCh. At least 2 lung slices from each mouse were evaluated. Inhibitor and DMSO treatments were performed from serial slices obtained from the same mouse.
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