For the migration assays, 105 cells in 100 μl of serum-free medium were placed into the upper chamber of a transwell insert (BD Biosciences). For the invasion assays, 105 cells in 100 μl serum-free medium were placed into the upper chamber of a transwell insert coated with Matrigel (BD Biosciences). Medium supplemented with 10% FBS (600 μl) was added to the lower chamber. The transwell plates were incubated at 37 °C in 5% CO2 for 12–48 h. The cells remaining on the upper membrane were removed with cotton wool. The cells that had migrated or invaded through the membrane were fixed with 4% paraformaldehyde, stained with crystal violet for 10 min, imaged and counted. Experiments were independently repeated three times.
Transwell insert
Manufactured by BD
Sourced in United States, United Kingdom
Transwell inserts are a type of cell culture equipment used to study cell migration, permeability, and co-culture experiments. They consist of a porous membrane that separates two compartments, allowing the passage of cells, molecules, and other materials between the upper and lower chambers.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (93 mentions)
Transwell insert, by Corning (9 mentions)
Inverted microscope, by Olympus (8 mentions)
Crystal violet, by Merck Group (7 mentions)
Matrigel, by Corning (6 mentions)
Axioplan 2 immunofluorescent microscope, by Zeiss (5 mentions)
Crystal violet, by Beyotime (4 mentions)
Crystal violet, by Sangon (4 mentions)
Crystal violet, by Solarbio (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Biocoat matrigel invasion chamber, by BD (3 mentions)
Growth factor reduced matrigel, by BD (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Matrigel basement membrane matrix, by Corning (3 mentions)
Matrigel basement membrane matrix, by BD (3 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (3 mentions)
Collagenase, by Merck Group (3 mentions)
Dnase, by Takara Bio (3 mentions)
Type 1 collagen, by BD (3 mentions)
Matrigel invasion chamber, by BD (2 mentions)
350 protocols using transwell insert
1
Cell Migration and Invasion Assays
2
Cell Migration and Invasion Assay
3
Transwell Assay for Cell Migration and Invasion
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Cell migration and invasion were assessed using a transwell assay. For migration assays, MGC803 and BGC823 cells were harvested; then 1 × 105 cells suspended in 100 μL of serum-free medium were placed in a transwell insert (pore size, 8 μm; BD Biosciences, San Jose, CA, USA). The lower chamber was filled with 600 μL medium containing 10 % FBS. For invasion assays, cells were suspended as in the migration assay, then placed into a transwell insert pre-coated with Matrigel (BD Biosciences, Bedford, MA, USA). After incubating cells for 24 h at 37 °C and gently removing the cells in the upper chamber with a cotton swab, the cells on the underside of the membrane were fixed with 4 % paraformaldehyde for 15 min, stained with 0.1 % crystal violet in 20 % ethanol, and counted in five randomly selected fields using phase contrast microscopy. Cells were imaged at 200× magnification using a Olympus microscope (Hamburg, Germany). Five independent fields per well were imaged. Each assay was performed in triplicate.
4
Cell Migration and Invasion Assays
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For migration assays, a total of 5×104 cells treated with the specified drugs were seeded into transwell inserts (BD Biosciences) and cultured for an additional 24 h. For invasion assays 105 cells treated with the specified drugs were seeded into a thick layer of Matrigel in a transwell insert. Cells that adhered to the lower surface of the filter were washed with phosphate-buffered saline, stained with 0.1% crystal violet solution, then examined via light microscopy at 100× magnification (Carl Zeiss, Inc.).
5
Transwell Migration and Invasion Assay
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HeLa and CaSki cells were seeded in DMEM culture in the top chamber of each Transwell insert (BD Biosciences, San Jose, CA, USA) at 1.0×105 cells/well with 8.0-mm pores for the migration assay. For the invasion assay, 2.0×105 cells were cultured in DMEM culture in the upper chamber of a Transwell insert (BD Biosciences) at 37°C for 24 h that was pre-coated with 0.2% Matrigel (Oscient Pharmaceuticals Corporation, Waltham, MA, USA) at 37°C. As a chemoattractant, 10% fetal bovine serum was added to the DMEM culture medium in the lower chamber. Then, Ad-p65 or Ad-NC/Ad-USF1 or Ad-NC was placed into the lower chamber at 37°C for 24 h. Following incubation for 24 h at 37°C, remaining cells in the upper chamber were removed using cotton swabs, and those which had migrated or invaded through the membrane were stained with a dye solution containing 20% methanol and 0.1% crystal violet at 37°C for 15 min. Cells were subsequently imaged under a light microscope (magnification, ×40; Olympus Corporation, Tokyo, Japan) and 10 individual fields were randomly chosen and counted per insert. The results are presented as the mean of three separate experiments.
6
Directional Cholesterol Transport in Trophoblast Monolayers
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Our final goal was to determine the directionality of the apoA-1 mediated cholesterol efflux using STB monolayers grown on Transwell® inserts (Falcon, Durham, NC, USA) consisting of apical (maternal-like) and basal (fetal-like) compartments. For determining 3H-cholesterol uptake and efflux, 12 well Transwell® inserts with 0.4 μm pore PET membrane were used which were coated with Matrigel (BD Biosciences, Bedford, MA, USA) at a concentration of 25 µg/cm2 prior to the experiment. The procedures used for 3H-cholesterol loading, uptake, and efflux determination were similar to those described for conventional plates with the exception that 1 × 106 cells/cm2 were seeded. The cholesterol transport studies were performed on day 7, after having validated both the tightness and barrier integrity of the STB monolayer, as previously described [46 (link)]. In brief, trans-epithelial electrical resistance (TEER) was measured daily using a Millicell ERS-2 Volt-Ohm Meter (Millipore, Bedford, MA, USA), as previously described [46 (link)]. The TEER value was determined on the Matrigel-coated inserts in the presence or absence of trophoblast cells. In addition, the monolayer permeability properties were evaluated by measuring daily the passage of the fluorescence dye Lucifer yellow (LY; Sigma, Saint Louis, MO, USA) across the trophoblast monolayer as previously described in detail [46 (link)].
7
Transwell Invasion Assay Protocol
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A total of 5×104 cells were suspended in 200 µl serum-free RPMI-1640 medium and seeded into the upper chambers of Transwell inserts (BD Biosciences). The Transwell inserts had been coated with 30 µl Matrigel (BD Biosciences) and incubated in the incubator for 4 h. A total of 500 µl RPMI-1640 medium containing 10% fetal calf serum (FCS; Gibco; Thermo Fisher Scientific, Inc.), used as the chemo-attractant, were added to the lower wells. Following incubation for 16 h, the chambers were fixed with 4% paraformaldehyde for 20 min at room temperature and stained with crystal violet (Beyotime Institute of Biotechnology) for 30 min at room temperature. Cells on the lower membranes were counted in five randomly selected fields (magnification, ×200) under a light microscope.
8
Cell Migration and Invasion Assay
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For migration assays, transfected cells were trypsinized at 48 h post-transfection, rinsed with phosphate buffer solution, collected via centrifugation and resuspended in FBS-free medium. The upper chambers of transwell inserts (BD Biosciences) were loaded with 200 μL cell suspension containing 5 × 104 cells, while basal medium supplemented with 10% FBS was added to the lower chambers. After 24 h, the non-migrated cells were removed with a cotton swab, while the migrated cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Subsequent to extensive washing, the migrated cells were imaged with an inverted microscope (IX31; Olympus Corporation, Tokyo, Japan), after which six visual fields were randomly selected and the average number of migrated cells was calculated. Transwell invasion assay was conducted using the same experimental protocols as the migration assay, with the exception that the transwell inserts were precoated with Matrigel (BD Biosciences).
9
Transwell Chemotactic Migration and Invasion
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Chemotactic migration and invasion were measured using the Transwell assay, as previously described [40 (link)]. In brief, for migration assay, HCV-infected and non-infected Huh7.5 cells were seeded in transwell inserts (BD, San Jose, CA, USA) at 80–90% confluence and allowed to invade for 24 h towards the medium. At the end of the incubation period, cells that were invaded through the membrane of the insert were stained with crystal violet and counted. Invasion assay was performed as above, but the transwell inserts were coated with Matrigel (BD) before the cells were seeded.
10
Evaluating Invasive Potential of NSCLC Cells
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We explored the invasive ability of NSCLC cells using Transwell inserts (BD Biosciences). We added 100 µL of Matrigel (BD Biosciences) into the inner sides of the Transwell inserts and performed polymerization by incubating them at 37°C for 2 h. Next, we loaded the upper chambers with 100 μL of FBS-free culture medium containing 4 × 104 of transfected cells, and plated 600 μL of culture medium supplemented with 10% FBS into the lower chambers. We allowed the NSCLC cells to penetrate the pores in the submembrane surface for 24 h, then fixed them with 100% methanol, and stained them with 0.1% crystal violet. The cells that invaded the lower chambers were counted in five randomly chosen regions (in each well) under an inverted microscope. For the migration assay, we did not apply Matrigel, but the remaining steps were the same as those in the Matrigel invasion assay.
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