Migration and invasion of HCC cells was examined using the transwell assay with polyethylene terephthalate membranes (24-well inserts, 8.0 μm, Corning). For the migration assay, 150 μL cell suspension containing 2 × 105 cells was loaded into the upper well coated without Matrigel (BD Biosciences). For the invasion assay, the Matrigel (BD Biosciences) was added. Next, 500 μL DMEM medium with 10% FBS was placed into the bottom of the well. Seventy-two hours later, invasive cells were stained with 0.1% crystal violet. Four fields were randomly selected and the cells counted. The experiments were repeated thrice and the data are presented as the mean ± SD.
24 well inserts 8.0 μm
Manufactured by Corning
The 24-well inserts with 8.0 μm pore size provide a platform for applications that require cell culture in a multiwell format. These inserts are designed to fit standard 24-well cell culture plates and enable the study of cellular processes, such as migration and invasion, in a controlled environment.
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6 protocols using 24 well inserts 8.0 μm
1
Transwell Assay for Cell Migration and Invasion
2
Transwell Migration and Invasion Assays
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Transwell migration and invasion assays were performed using transwell with polyethylene terephthalate membranes (24-well inserts, 8.0 μm, Corning)15 (link). For migration assay, 200 μL cell suspensions contained 5 × 104 cells was loaded into the upper chamber of a transwell. Next, 600 μL DMEM medium with 10% FBS was placed into the bottom of the well as a source of chemo-attractants. 24 h later, the cells on the lower surface of the insert were fixed with methanol and staining with 0.5% crystal violet. For transwell invasion assay, a similar procedure was performed as the above description except that 1 × 105 cells was loaded into upper chamber pre-coated with matrigel (BD Biosciences, CA, USA). Staining cells were visualized and photographed using a CKX41 microscope (Olympus, Japan). Randomly selected five fields and counted the cells, experiments were repeated three times and the data are presented as the means ± SD.
3
Evaluating BCa Cell Migration and Invasion
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The polyethylene membranes (24-well inserts; 8.0 μm; Corning, Inc.) were utilized to detect the migration and invasion abilities of BCa cells. Chambers precoated with 50 µL Matrigel (BD Biosciences) at 37°C for 1 h were used for invasion assays, while uncoated chambers were utilized for migration assays. Cell suspensions containing 1×105 cells in 100 μL FBS-free DMEM were seeded in the upper chamber. Meanwhile, the lower chamber was covered with 500 μL DMEM supplemented with 10% FBS. Cells were cultured at 5% CO2 and 37°C for 48 hours. After 48 hours, cells that have remained in the upper membranes were gently removed using a cotton swab; while cells that have migrated or invaded the bottom of the membrane were fixed with polyoxymethylene at room temperature for 20 min and then stained with 0.5% crystal violet at room temperature for 20 min. Cells were counted in 5 randomly selected fields under a light microscope (Leica Microsystems GmbH, Wetzlar, Germany).
4
Osteosarcoma Cell Migration and Invasion
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The polyethylene membranes (24-well inserts; 8.0 μm; Corning, Inc.) were utilized to detect the migration and invasion abilities of osteosarcoma cells. For cell invasion assay, the upper surface of the membrane was coated with 50 µL Matrigel, incubated at 37°C for 5 h, while the membrane was uncoated during cell migration assay. The mixed cell suspension solution (1 × 105 cells in a volume of 150 μL serum-free medium) was added to the upper chamber, and then a total of 500 μL culture medium containing 10% FBS was added to the lower chamber. Cells were cultured at 5% CO2 and 37°C for 48 hours. The cells on the upper layer were wiped off with a cotton swab. Next, the chamber was fixed with methanol at room temperature for 10 min, stained with 0.1% crystal violet solution for 10 min. Five visual fields were randomly selected for image capture, and the cells were counted under a light microscope (Olympus Corporation) at ×400 magnification.
5
Transwell Migration Assay Protocol
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Transwell assay was performed in 24-well inserts (8.0 μm; Corning). Briefly, 5 × 10 5 cells suspended in 200 μl serum-free medium were loaded on the top, and 500 μl complete medium was placed into the bottom side. 48 h later, the migrated cells were stained with 0.1% crystal violet. Images were captured at room temperature using Echo Revolve Hybrid Microscope (Echo) using a 10× objective at brightfield mode. Cells were counted using ImageJ software and the data were presented as the mean ± SEM., calculated from four randomly selected fields.
6
Quantifying NSCLC Cell Migration and Invasion
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The migration and invasion abilities of NSCLC cells were detected by polyethylene membranes (24-well inserts; 8.0 μm; Corning, Inc.). The chambers precoated with 50 µL Matrigel (BD Biosciences) at 37°C for 1 h were used for invasion assay, and those uncoated chambers were used for migration assay. The upper chamber was seeded with cell suspension containing 1×105 cells in 100 μL FBS-free RPMI 1640, and the lower chamber was covered with 500 μL RPMI 1640 supplemented with 10% FBS. Cells were cultured at 5% CO2 and 37°C for 48 h. Cells that remained in the upper membrane were gently removed using a cotton swab, and cells that had migrated or invaded the bottom of the membrane were fixed with polyoxymethylene at room temperature for 20 min and then stained with 0.5% crystal violet at room temperature for 20 min. Cells were counted in five randomly selected fields under the light microscope (Leica Microsystems GmbH, Wetzlar, Germany).
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