15,000 myoblasts (mdx or w/t) were seeded into a 24-well cell culture plate and grown for 48 h in the culture medium. Time-lapse movies were generated by multiple well areas per each cell type being photographed every 15 min for 5 h 45 min using the inverted DMI6000 microscope (Leica Microsystems GmbH) equipped with an environmental chamber (PeCon GmbH) and a DFC350FXR2 CCD camera (Leica Microsystems GmbH). Both the bright field and the DIC Nomarski contrast using HC PL APO 10×/0.40 dry objective (Leica Microsystems GmbH) were captured. Acquired time-lapse movies were exported to TIFF format and aligned to compensate for possible drifts using an ImageJ plugin. Subsequently, at least 30 cells per each experimental condition were tracked semi-automatically in the time-lapse movies using the Track Objects plug-in in Leica MM AF powered by MetaMorph® software (Leica Microsystems GmbH). Cells dividing or colliding with other cells were excluded from the analysis. The statistical significance was evaluated as described below.
Mm af
Manufactured by Leica
The Leica MM AF is a high-precision, automated microscope system designed for a wide range of microscopy applications. It features advanced autofocus capabilities and can be configured with a variety of objective lenses and accessories to accommodate different imaging needs.
Automatically generated - may contain errors
Lab products found in correlation
Dmi6000 inverted microscope, by Leica (2 mentions)
Dfc350fxr2 ccd camera, by Leica (2 mentions)
Metamorph software, by Leica (2 mentions)
Metamorph software, by Molecular Devices (2 mentions)
Matrigel, by Thermo Fisher Scientific (2 mentions)
Jem 100c, by JEOL (1 mentions)
Hf 3300, by Hitachi (1 mentions)
Matlab r2016b, by MathWorks (1 mentions)
Hc pl apo 10x 0.40 dry objective, by Leica (1 mentions)
Fibronectin, by Merck Group (1 mentions)
5 protocols using mm af
1
Time-Lapse Microscopy of mdx and WT Myoblasts
2
Ultrastructural Analysis of Auditory Pathway
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Following pentobarbital injection (100 mg/kg), animals to have EM examination of their brains underwent transcardiac perfusion with heparinized 0.9% NaCl, followed by 500 mL of 4% paraphormaldehyde and 2.5% glutaraldehyde in 0. 1 M phosphate buffer (PB), pH 7.4, at a perfusion pressure 120 mm Hg. The brains were removed from skull and placed in the same fixative overnight. The right hemispheric tissue blocks containing the inferior colliculi (IC) and the medial geniculate body (MGB)were cut into 400 micron-thick coronal slices. Slices were washed in cold 0.1 M PB and kept in 2.5% glutaraldehyde in 0.1 M PB until processing; when processing, the slices were washed in cold PB, post-fixed in 1% osmium tetroxide in cold PB for 2 h and again washed in 0.1 M PB. The IC and GMB were identified with an optical microscope Leica MM AF, cut out from the coronal slices, dehydrated in graded series of ethanol and acetone, and embedded in araldite. Blocks were trimmed and 70–75 nm thick sections were cut with an ultra-microtome Reichert, picked up on 200-mesh copper grids, double-stained with uranyl-acetate and lead-citrate, and examined with a JEM 100 C (JEOL, Japan), HF 3300 (Hitachi, Japan) and Tesla BS 500, Czechoslovakia) transmission electron microscopes. For each case, 120 sections were observed.
3
Random Migration Assay for Myoblasts
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To perform random migration assays 15,000 myoblasts (mdx and w/t) were seeded into 24-well cell culture plate and grown for 30 h in an appropriate culture medium. Then, multiple different well areas per each cell type were photographed in the bright field or DIC Nomarski contrast using HC PL APO 10x/0.40 Dry objective (Leica Microsystems GmbH) every 15 min for 6 h on inverted DMI6000 microscope (Leica Microsystems GmbH) equipped with DFC350FXR2 CCD camera (Leica Microsystems GmbH) and an environmental chamber (PeCon GmbH). Acquired time-lapse movies were exported to TIFF format and aligned to compensate possible drift using an ImageJ plugin. Subsequently, at least 15 cells of each experimental condition were tracked semi-automatically in the time-lapse movies using Track Objects plug-in in Leica MM AF powered by MetaMorph® software (Leica Microsystems GmbH). Cells dividing or colliding with other cells were excluded from the analysis. Obtained cell coordinates were subsequently imported into Matlab R2016b software (MathWorks, Inc.) and the movement parameters were calculated using a Matlab script. Each experiment was performed at least in triplicate. Statistical analysis was performed using Student's t test.
4
2D Random Cell Migration Assay
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We dispensed 40,000 or 50,000 cells per well into twelve-well plates coated with Matrigel (Thermo Fisher Scientific) or 40 ng/μl of fibronectin (#F1141; Sigma-Aldrich) (for 12S2 or MeWo/A375M, respectively). Two-dimensional (2D) random cell migration was monitored by time-lapse video microscopy under bright white light, with an inverted phase-contrast microscope (Leica MM AF) equipped with a cell culture chamber (37°C, humidified atmosphere containing 5% CO2), an x–y–z stage controller, and a charge-coupled device CoolSnap camera (Photometrics). Images were acquired at 8-min intervals during 16 h, with the Metamorph software (Molecular Devices). Movies were reconstructed with the ImageJ software (http://rsbweb.nih.gov/ij/ ). Cells were tracked manually by using the center of the nucleus as guide and parameters were calculated with ImageJ Manual Tracking plug-in. Individually tracked cells were chosen to be alive by eye monitoring over the entire duration of the movie. Briefly, the manual tracking plug-in is recording x and y positions of each cells tracked and then generate statistical values as the mean overall velocity (in μm/h), total distance travelled (in μm) or % pausing (which is the cumulative fraction of time of the total duration of the movie where the cells are not changing position).
5
2D Random Cell Migration Monitoring
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We dispensed 50,000 cells per well into twelve-well plates coated with Matrigel (FisherScientific). Two-Dimensional (2D) random cell migration was monitored by time-lapse video microscopy under bright white light, with an inverted phase contrast microscope (Leica MM AF) equipped with a cell culture chamber (37°C, humidified atmosphere containing 5% CO2), an x-y-z stage controller and a charge-coupled device (CCD) CoolSnap camera (Photometrics). Images were acquired at 8-minute intervals during 16 hours, with the Metamorph software (Molecular Devices). Movies were reconstructed with the ImageJ software (http:// rsbweb.nih.gov/ij/). Cells were tracked manually and parameters were calculated with ImageJ Manual Tracking plug-in.
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