For the migration assays, 5×104 cells were added into the upper chamber of the insert (BD Bioscience, 8-µm pore size). For the invasion assays, 1×105 cells were added into the upper chamber of the insert precoated with Matrigel (BD Bioscience). In both assays, cells were plated in medium without serum, and medium containing 10% FBS in the lower chamber served as chemoattractant. After several hours of incubation, the cells that did not migrate or invade through the pores were carefully wiped out with cotton wool. Then the inserts were stained with 20% methanol and 0.2% crystal violet, imaged, and counted with an IX71 inverted microscope (Olympus).
Upper chamber of the insert
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The upper chamber of the insert is a component used in various laboratory equipment. Its core function is to provide a controlled environment for cell culture and related experiments.
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3 protocols using upper chamber of the insert
1
Cell Migration and Invasion Assays
2
Cell Migration Assay Protocol
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Cells were harvested and re-suspended in serum-free DMEM medium. For the migration assay, 2.5 × 104 cells were added into the upper chamber of the insert (BD Bioscience, 8 μm pore size). Cells were plated in medium without serum, and medium containing 20% FBS in the lower chamber served as the chemoattractant. After 6 h of incubation, cells were fixed with 4% formaldehyde and stained with crystal violet staining solution, and cells on the upper side of the insert were removed with a cotton swab. The migratory capacity was evaluated as the total number of cells on the lower surface of the membrane, as determined by microscopy.
3
Cell Migration Assay Protocol
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Fadu cells were grown to 50-70% con uence. We added the cells into the upper chamber of the insert (BD Bioscience, 8-µm pore size) for the migration assays. We incubated the cells in medium without serum, yet with 10% FBS in the lower chamber for chemo attractant in both assays. After a few hours of incubation, we wiped out the cells which did not migrate through the pores carefully with cotton wool. Finally, we stained the cells with 0.2% crystal violet and 20% methanol, and counted the imaged inserts.
Statistical analysis SPSS 21.0 software package (SPSS, Inc., Chicago, IL, USA) was applied for statistical calculations. The evaluation of the differences between two groups was conducted using the Student t-test. And one-way analysis of variance was applied to analyze the comparison of the means greater than or equal to three groups. Data were presented as the mean ± standard error of the mean (abbreviated as SEM). P < 0.05 was de ned as statistically signi cant.
Statistical analysis SPSS 21.0 software package (SPSS, Inc., Chicago, IL, USA) was applied for statistical calculations. The evaluation of the differences between two groups was conducted using the Student t-test. And one-way analysis of variance was applied to analyze the comparison of the means greater than or equal to three groups. Data were presented as the mean ± standard error of the mean (abbreviated as SEM). P < 0.05 was de ned as statistically signi cant.
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