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.
Hc pl apo 10x 0.40 dry objective
Manufactured by Leica
The HC PL APO 10x/0.40 Dry objective is a high-quality microscope objective lens produced by Leica. It features a magnification of 10x and a numerical aperture of 0.40, making it suitable for a variety of microscopy applications. The lens is designed for use in dry conditions and provides consistent, reliable performance.
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2 protocols using hc pl apo 10x 0.40 dry objective
1
Random Migration Assay for Myoblasts
2
Time-lapse Imaging of Myoblast Dynamics
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15,000 myoblasts (mdx and w/t) were seeded into 24-well cell culture plate and grown for 48 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 5 h 45 min. 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 30 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, which has been performed as described below.
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