Primary human PASMCs were isolated from donor lungs as previously described (42 (link)) (gift of Suzy Comhair and Serpil Erzurum, Cleveland Clinic Foundation) and seeded into 24-well plates at a density of 1.5 × 104 cells per well in human smooth muscle cell (SMC) basal medium supplemented with human SMC growth supplement (both Cell Application, Inc.). At 30% confluence, cells were transfected with 50 ng of control or β-arrestin-1 and β-arrestin-2 ON-TARGETplus siRNA pools (Dharmacon, Inc.). When the cells reached 100% confluency, they were serum starved for 4 hours with 2 hours of PTx (200 ng/ml) treatment. Scratches were made using small pipette tips. Cells were washed using Dulbecco’s PBS and stimulated with basal SMC medium containing 1 μM AngII (Millipore Sigma) or 10 μM TRV120023 (Genscript USA Inc.). The wound closure was monitored using a live-cell station Zeiss Axio Observer microscope (Carl Zeiss, Thornwood, NY). The images were captured in real time at 0 hours and for every hour up to 16 hours. The initial edges of the scratch at the 0-hour time point were marked, and the migrated distance for 12 to 16 hours afterwards was measured using MetaMorph Premier software (Molecular Devices, San Jose, CA) at the Duke Light Microscopy Core Facility (Durham, NC).
Metamorph premier software
Manufactured by Molecular Devices
Sourced in United States, Germany, Japan
MetaMorph Premier software is a comprehensive image acquisition and analysis platform designed for microscopy and cell biology applications. It provides a robust set of tools for controlling a wide range of microscope hardware, capturing high-quality images, and performing advanced image analysis.
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Lab products found in correlation
Ang 2, by Merck Group (1 mentions)
Axio observer microscope, by Zeiss (1 mentions)
Trv120023, by GenScript (1 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Sigmaplot, by Grafiti LLC (1 mentions)
Dmi6000b microscope, by Leica (1 mentions)
Csu x1 spinning disk unit, by Yokogawa (1 mentions)
Axio imager z1 microscope, by Zeiss (1 mentions)
Metamorph premier, by Molecular Devices (1 mentions)
100 w highpressure mercury burner, by Olympus (1 mentions)
Orca 2 ccd camera, by Hamamatsu Photonics (1 mentions)
Disc spinning unit, by Olympus (1 mentions)
Lambda xl light source, by Sutter Instruments (1 mentions)
Sit camera c10600, by Hamamatsu Photonics (1 mentions)
Lamba 10b optical filter changer, by Sutter Instruments (1 mentions)
Forskolin, by Merck Group (1 mentions)
1260 infinity hplc system, by Agilent Technologies (1 mentions)
6 protocols using metamorph premier software
1
Wound Healing Assay for Primary Human PASMCs
2
Quantifying Vessel-like Structure Formation
3
Confocal Microscopy of Skin Sections
4
Visualizing Mitochondrial Redox State in C. elegans
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After the acute swim exercise on day 4, we fixed C. elegans at different time points in 2% paraformaldehyde/phosphate buffered saline for 30 min at room temperature and then stored them at 4 °C in M9 buffer until imaging. We performed confocal imaging with a CSU-X1 spinning disk unit (Yokogawa, Sugar Land, TX, USA) mounted to an Axio Imager Z1 microscope (Zeiss, Oberkochen, Germany) using MetaMorph Premier software (Molecular Devices, Sunnyvale, CA, USA).
For Pmyo-3mito-roGFP transgenic animals (KWN122 strain), we imaged body wall muscle cells at both 405 nm and 488 nm excitation with a 525/50 nm emission filter. We performed image analysis in ImageJ by manual selection of 40–50 mitochondrial regions per image, quantitating the mean fluorescence intensities of the exact same mitochondrial regions in both 405 nm and 488 nm images, and calculating the 405/488 ratio for each region. For each animal we generated a single 405/488 ratio value, which corresponded to the average of all 40–50 of the 405/488 ratios.
For tissue-specific PLIN1::GFP transgenic animals (XD3971, XD2458, and XD1875 strains), we imaged intestine, hypodermis, or body wall muscle at 488 nm excitation with a 525/50 nm emission filter. We quantitated the number of lipid droplets manually in ImageJ and normalized to the area analyzed in each image.
For Pmyo-3mito-roGFP transgenic animals (KWN122 strain), we imaged body wall muscle cells at both 405 nm and 488 nm excitation with a 525/50 nm emission filter. We performed image analysis in ImageJ by manual selection of 40–50 mitochondrial regions per image, quantitating the mean fluorescence intensities of the exact same mitochondrial regions in both 405 nm and 488 nm images, and calculating the 405/488 ratio for each region. For each animal we generated a single 405/488 ratio value, which corresponded to the average of all 40–50 of the 405/488 ratios.
For tissue-specific PLIN1::GFP transgenic animals (XD3971, XD2458, and XD1875 strains), we imaged intestine, hypodermis, or body wall muscle at 488 nm excitation with a 525/50 nm emission filter. We quantitated the number of lipid droplets manually in ImageJ and normalized to the area analyzed in each image.
5
Quantifying Angiogenic Tube Formation
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Vessel formation was assessed at the aforementioned time points. Fluorescent images were captured utilizing an Olympus IX81 equipped with Disc Spinning Unit and a 100 W high-pressure mercury burner (Olympus America, Center Valley, PA), a Hamamatsu Orca II CCD camera (Hamamatsu Photonics, K.K., Hamamatsu City, Japan), and Metamorph Premier software (Molecular Devices, Sunnyvale, CA). Imaged beads were chosen at random provided that vessels emanating from a given bead did not form anastomoses with vessels from adjacent beads. Images from at least 30 beads per condition were captured over three separate trials at low magnification (4×) for each independent experiment and processed using the Angiogenesis Tube Formation module in Metamorph Premier (Molecular Devices). Each image was segmented and analyzed based on any tube-like pattern that falls within a specified minimum and maximum width of each segment above a contrast threshold. The total network length, the number of branch points, and number of segments were quantified.
6
Calcium Imaging of Odorant Responses in Dissociated Olfactory Sensory Neurons
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After being washed with fresh Ringer’s solution, the coverslips were mounted on a recording chamber. Imaging was carried out at room temperature on an inverted fluorescence microscope (IMT-Olympus) equipped with an SIT camera (C10600, Hamamatsu Photonics), a Lambda XL light source (Sutter Instrument), and Lamba-10B optical filter changer (Sutter Instrument). Using a 1260 Infinity HPLC system (Agilent Technologies), the dissociated OSNs were stimulated with the odorants in random order. A final stimulation with a 10 μM forskolin (Sigma-Aldrich) solution was made to assess the viability of the OSNs. Recordings were made at 490-nm excitation and 520-nm emission. Images were taken every 4 s, and there was a 4-min delay between stimulations. The images were then computed using MetaMorph Premier software (Molecular Devices LLC), and the cells were manually counted. Cells exhibiting an intensity increase of at least 10% ΔF/F0 amplitude between 8 and 12 frames after the odorant injection were considered responsive cells.
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