The migration and invasion abilities were determined using a Transwell apparatus without or with Matrigel-coated membranes (BD Biosciences, Franklin Lakes, NJ). The migration and invasion assay procedure was detailed in a previous study [11 (link)]. The total number of attached cells was counted. All experiments were performed in triplicate. Single cell migration assay was performed as following description. Mock and stably SPANXA-expressing CL1-5 cells were seeded 5000 cells/per well in 6-well culture plate for overnight attachment. Wash wells with PBS twice and replace fresh medium. Set up the focus and sites for 25 position /per well, and take the picture every 1 hour in 24 hours. The high content screening software MetaMorph (Molecular Devices, Sunnyvale, CA) was used to analyze every single cell tracking and mean velocity.
Transwell apparatus
Manufactured by BD
Sourced in United States
The Transwell apparatus is a cell culture insert system designed for permeability and transport studies. It consists of a permeable membrane that separates two chambers, allowing the user to monitor the movement of cells, molecules, or other substances between the compartments.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem reduced serum media, by Thermo Fisher Scientific (2 mentions)
Optical microscope, by Olympus (2 mentions)
Matrigel, by BD (2 mentions)
Matrigel coated membrane, by BD (2 mentions)
Metamorph, by Molecular Devices (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Matrigel, by Merck Group (1 mentions)
Fv1000, by Olympus (1 mentions)
Matrigel coated, by BD (1 mentions)
Transwell apparatus, by Corning (1 mentions)
Biocoat matrigel, by BD (1 mentions)
Fetal bovine serum (fbs), by Solarbio (1 mentions)
Prism 7, by GraphPad (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
21 protocols using transwell apparatus
1
Transwell Migration and Invasion Assay
2
Cell Invasion Assay Protocol
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A total of 3 × 104 cells were transferred into the upper chambers of a Transwell apparatus (8 μm, BD Biosciences, CA, USA). The bottom chamber was filled with a complete medium supplemented with 10% FBS. After incubation for 48 h, cells that did not invade through the membrane were swept away. Then, the cells were fixed with 20% methanol and stained with 0.2% crystal violet. Cells invading into the bottom chamber per field were counted under an inverted microscope.
3
Transwell Invasion Assay
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A total of 3 × 104 cells were added to the upper chamber of the Transwell apparatus (8-μm, BD Biosciences, CA, USA) with a Matrigel-coated membrane (BD Bioscience, CA, USA) for the invasion assay. The bottom chamber was filled with complete medium supplemented with 10 % FBS. After 48 h, the cells that did not invade through the membrane were swept. The remaining cells were then fixed with 20 % methanol and stained with 0.2 % crystal violet. The cells that invaded into the bottom chamber per field were counted.
4
Cell Invasion Assay with Transwell
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Cells (3×104) were transferred to the upper chamber of a Transwell apparatus (8-µm; BD Biosciences). As a chemoattractant, the bottom chamber was filled with complete medium supplemented with 10% FBS. After 48 h of incubation, the cells that did not invade through the membrane were wiped. The cells were then fixed with 20% methanol and stained with 0.2% crystal violet. Cells invading the bottom chamber per field were counted under an inverted microscope (Olympus IX71).
5
Cell Invasion Assay with Transwell
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After transfection for 24 h, cells in serum-free media were seeded into the upper chamber of a Transwell apparatus (BD Biosciences). for invasion assays with Matrigel (Sigma-Aldrich, St. Louis, MO, USA). The lower chambers were filled with media containing 10% FBS. After 24 h of incubation at 37°C in a 5% CO2 atmosphere, the cells which had invaded through the membrane were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet (Sigma-Aldrich). The cells on the lower surface were photographed (Olympus; IX73) and three random fields were counted using a confocal laser-scanning microscope (Olympus FV1000). Three independent experiments were performed.
6
Transwell Cell Invasion Assay
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In total, 3 × 104 cells were transferred to the upper chamber of a Transwell apparatus (8-μm; BD Biosciences, CA, USA). As a chemoattractant, the bottom chamber was filled with complete medium supplemented with 10% FBS. After 48 h of incubation, the cells that did not invade through the membrane were collected. The cells were then fixed with 20% methanol and stained with 0.2% crystal violet. Cells invaded into the bottom chamber per field were counted under an inverted microscope.
7
Cell Colony Formation and Migration Assays
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For cell colony formation assay, A549 or H1792 cells were transfected with siRNA or pcDNA for 24 h and seeded into 6-well plates (500/well). After 1 week, we fixed formed colonies and stained with 0.1% crystal violet in 20% methanol, and counted colonies consisting of at least 50 cells. For the migration assay, we resuspended 5 × 105 cells in Opti-MEM Reduced Serum Media (Invitrogen) and seeded into the upper chamber of a transwell apparatus (8.0 μm, BD Biosciences), and added complete medium to the bottom chamber to provide chemoattractants for migration. To count migrated cells, we captured images of stained cells in five random fields by using an optical microscope (Olympus, Japan) and counted samples in triplicate.
8
Transwell Invasion Assay Protocol
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Cells (3×104) were transferred to the upper chamber of a Transwell apparatus (8-µm; BD Biosciences) in serum-free medium. Before inoculating the cells, Matrigel (BD Biosciences) was diluted (1:8) and added to the upper chamber for 30 min at 37°C. As a chemoattractant, the bottom chamber was filled with complete medium supplemented with 10% FBS. After 48 h of incubation at 37°C, the cells that did not invade through the membrane were removed. The cells were then fixed with 20% methanol at room temperature and stained with 0.2% crystal violet at 37°C for ~30 min. The cells invading the bottom chamber per field were counted under an optical light microscope (magnification, ×400; IX71; Olympus Corporation).
9
Transwell Assay for Cell Invasion
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Cell invasion ability was analyzed using a Transwell apparatus (BD Biosciences, Franklin Lakes, NJ, USA). MDA-MB-231 cells were plated at a density of 3×104 cells/well and incubated at 37°C with 150 ng/ml rHuPCSK6 in the upper chamber of the Transwell apparatus for 8 h. The upper and lower chambers were incubated with DMEM without FBS for 12 h, then DMEM containing 20% FBS was placed in the lower chamber for 48 h. Cells remaining in the upper chamber were removed with cotton swabs, and the cells which had invaded the lower chamber were stained with 10% Giemsa for 10 min at room temperature (cat no. G1015; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The number of invasive cells were counted in 5 random fields using a light microscope (Nikon Corporation, Tokyo, Japan; magnification, ×100).
10
Cell Migration Potential Assay
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The migration potential was measured using a transwell apparatus (8‐μm pore size, Corning, NY, USA). A 200 μL volume of cell suspension containing 100 000 transfected cells was plated in medium containing 1% FBS in the top chamber of a transwell apparatus with or without coated matrigel (BD Bioscience, Franklin Lakes, NJ, USA), while 700 μL of medium containing 20% FBS was placed in the lower chamber. After 36 h of incubation, cells were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 20 min. Cells on the undersides of the filters were photographed using a microscope (magnification 200×). At least three random fields in each well were imaged and analyzed.
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