Transwell inserts (Corning Incorporated, Corning, NY, USA) were used to detect cell migration and invasion. For the migration assay, cells (5×105 cells/ml) cultured in serum-free medium from each group were added to the upper chamber, and fetal bovine serum (Beijing Transgen Biotech Co., Ltd.) containing DMEM (Thermo Fisher Scientific, Inc.) was added to the lower chamber. All the cells were left to migrate for 24 h. The migrated cells were fixed in 95% alcohol, stained with 0.1% hexamethyl pararosaniline (Beyotime Institute of Biotechnology, Haimen, China) for 20 min at room temperature, and counted under an upright metallurgical microscope (magnification, ×100; Leica Microsystems GmbH, Wetzlar, Germany). The experiment was repeated 3 times for each group. For invasion detection, upper chambers of Transwell were specifically coated with Matrigel. The rates of cell migration and invasion in all groups were calculated.
Upright metallurgical microscope
Manufactured by Leica Microsystems
Sourced in China
The Upright metallurgical microscope is a scientific instrument designed for the examination and analysis of solid materials, such as metals, ceramics, and other opaque samples. It is used to observe the microstructure, composition, and surface features of these materials at high magnifications. The microscope is equipped with various objectives and illumination systems to provide clear and detailed images of the sample.
Automatically generated - may contain errors
2 protocols using upright metallurgical microscope
1
Transwell-based Cell Migration and Invasion Assay
2
Transwell Invasion Assay Protocol
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A total of 10 5 cells in serum-free medium were seeded into the upper chamber of a Transwell insert (8-µm pore size) coated with Matrigel ® (BD Biosciences, Franklin Lakes, NJ, USA). Then media containing 15% FBS were added to the lower chamber and following 48 h of culture, the cells remaining on the upper membrane were removed and cells that invaded to the lower membrane were fixed in 4% paraformaldehyde at room temperature for 10 min and stained with 0.1% crystal violet at room temperature for 10 min and imaged. The cell numbers were calculated under an upright metallurgical microscope (Leica Microsystems GmbH) at 100x magnification.
Statistical analysis. In vitro results were presented as the mean ± standard error of the mean from at least three independent experiments. Mann-Whitney-U test, Spearman rank correlation coefficient test, Student's t test, analysis of variance and multiple comparison between the groups using the S-N-K method were performed using SPSS software (version 21.0; IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.
Statistical analysis. In vitro results were presented as the mean ± standard error of the mean from at least three independent experiments. Mann-Whitney-U test, Spearman rank correlation coefficient test, Student's t test, analysis of variance and multiple comparison between the groups using the S-N-K method were performed using SPSS software (version 21.0; IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.
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