Transwell assay was performed using transwell migration chambers (#3422, Costar, Cambridge, MA, USA) or invasion chambers (#354480, Costar). Briefly, ESCC cells re-suspended in cell medium (200 μL) were inoculated into apical chambers after transfection for 24 h. Also, 600 μL medium containing 10% FBS (Thermo Fisher Scientific) was added to basolateral chambers. After culture for a while (24 h), the migrating and invasive cells were fixed with 4% paraformaldehyde (Santa Cruz Biotechnology) and stained with 0.1% crystal violet (Santa Cruz Biotechnology). The cells in 5 random fields were figured using a Nikon microscope (× 100, Nikon Eclipse E600, Nikon Instruments).
Invasion chamber
Manufactured by Corning
Sourced in United States
Invasion chambers are a type of lab equipment used to study the migratory and invasive properties of cells. These chambers provide a controlled environment to observe and measure the ability of cells to penetrate or 'invade' through a barrier, which is often composed of a layer of extracellular matrix proteins. The core function of invasion chambers is to facilitate the quantitative analysis of cell invasion, a crucial aspect of various biological processes and disease states.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Matrigel, by Corning (2 mentions)
Crystal violet, by Santa Cruz Biotechnology (1 mentions)
Transwell migration chamber, by Corning (1 mentions)
Paraformaldehyde (pfa), by Santa Cruz Biotechnology (1 mentions)
Eclipse e600, by Nikon (1 mentions)
Anti phosphomlc ser19, by Cell Signaling Technology (1 mentions)
Ilomastat, by Merck Group (1 mentions)
Type 1 collagen, by R&D Systems (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Matrigel matrix, by BD (1 mentions)
Zometa, by Novartis (1 mentions)
Dm il, by Leica (1 mentions)
Triton, by Merck Group (1 mentions)
Matrigel matrix, by Corning (1 mentions)
Transwell chambers with 8 μm pore membrane, by Corning (1 mentions)
24 well plate, by Corning (1 mentions)
Transwell assay kit, by Corning (1 mentions)
Phase contrast microscope, by Olympus (1 mentions)
100ul matrigel, by BD (1 mentions)
22 protocols using invasion chamber
1
Transwell Migration and Invasion Assay
2
Invasion Assay of MG63 Cells
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In vitro invasion was analyzed using 24 well Invasion Chambers (8 µm pore size; Costar, Corning, USA). For the invasion assay, the transwell chambers were pre-coated with 50 µL Matrigel (Corning, USA) for 3 h. MG63 cells were seeded into the upper chambers at the density of 1×104/100 µL in serum free medium, and the lower chambers were filled with 500 µL DMEM supplemented with 10% FBS. After incubating for 24 h, the cells were washed twice with PBS and fixed with 4% paraformaldehyde for 30 minutes at room temperature. The PET membranes were air-dried and stained with 0.1% crystal violet for 15 minutes. The non-invaded cells were wiped off, and the invaded cells were photographed and counted under an inverted microscope in five random fields per membrane. Both assays were performed in triplicate.
3
Invasion and Migration Assay Protocol
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Unless specified, all reagents were obtained from Sigma (St. Louis, MO, USA) and all the antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) except for antiphospho MLC (Ser 19) (Cell Signalling, Danvers, MA, USA). Matrigel Matrix was purchased from BD Biosciences (San Jose, CA, USA). ZOMETA (Zoledronic acid) was from Novartis (Basel, Switzerland). The invasion chambers were from Corning Costar (NY, USA). Ilomastat was from Chemicon International (Temecula, CA, USA). Type I collagen was from RD Systems (Minneapolis, MN, USA). Fluorescein isothiocyanate (FITC)-Phalloidin was from Molecular Probes (Eugene, OR, USA). Secondary antibodies conjugated with Alexa Fluor 488 and CellTraceTM Carboxyfluorescein succinimidyl ester (CFSE) were from Life Technologies Invitrogen (Carlsbad, CA, USA). CellTracker™ Orange (5-(and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine) (CMTMR) Dye was from Thermo Fisher Scientific (Waltham, MA, USA). Glutathione S-transferase (GST)-Rhotekin was from Peprotech (London, UK) .
4
Osteosarcoma Cell Invasion Assay
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Invasion chambers (Corning, Inc.) containing miR-218, YM155, or NC-miRNA were used to study the invasive capacity of the cells, which is the ability to degrade and migrate extracellular substrates beyond the basement membrane. Osteosarcoma cells (5×105 cells/well) were suspended in minimum essential medium (MEM) or DMEM, seeded into the Matrigel-coated upper chamber and incubated with MMC (0.25 µg/ml) in the presence of 10% fetal bovine serum. After 72 h of treatment, cells that had passed to the bottom of the chamber were fixed in 4% paraformaldehyde for 15 min, stained with Giemsa (24°C, 2 min) and counted under phase-contrast microscope Leica DM IL (Leica Microsystems GmbH) using ×20 objective and measurements were taken in 10 fields of view per chamber.
5
Invasion Assay for Cell Migration
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Invasion chambers (Corning, NY, United States) were also used to detect CMEC migration. We added 150 μl of cell FBS-free suspension (1 × 105 cells/ml) to the inserts and medium with different drug concentrations to the lower chamber. After the treatment, non-invasive cells in the upper chamber were removed with cotton swabs and invasive cells were stained with DAPI. We counted six random fields for each chamber (magnification, ×200) using a microscope (Leica Microsystems, Germany).
6
Matrigel Invasion Assay Protocol
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Matrigel Matrix (Corning, 354230) was diluted to final concentration of 300 μg/mL in cold coating buffer (0.01 M Tris (pH8.0), 0.7% NaCl) before being added to invasion chambers (Corning Cat, 353097) and left to set overnight at 37 °C. 2 × 104 cells were added to each chamber in serum free medium and 0.75 mL complete medium was added to the wells. Cells were allowed to invade 24 h in cell culture incubator. Invading cells were fixed and permeabilised with 4% PFA (Electron Microscopy Sciences, 15713-S) and 0.1% Triton (Sigma). Non-Invading cells were removed using a cotton swab. Cells were stained with Crystal violet solution. For all experiments three technical and three biological replicates were performed.
7
Cell Migration and Invasion Assay
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Cell migration and invasion assays were carried out using Transwell chambers with 8-μm pore membrane (Corning) and Invasion chambers (Corning), respectively, according to the manufacture’s instruction. Briefly, 2 × 104 MDA-MB-231 cells infected with pBabe-puro-empty (Empty) and pBabe-puro-GATA3 (GATA3) and serum-starved for 24 h were seeded into the upper chamber in 2% FBS-containing medium. 20% FBS-containing medium was added into the lower chamber. 24 or 48 h later, the medium and the cells remaining on the upper surface of the membrane were removed with cotton swabs. The cells on the lower surface of the membrane were fixed and stained with 0.1% crystal violet. The cells in five randomly chosen microscopic fields were counted and averaged.
8
Cell Migration and Invasion Assay
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The migration assay was conducted with transwell plates with 8μm pores (Corning Incorporated, Corning, NY, USA). Cell invasion assays were performed using invasion chambers (Corning Incorporated, Corning, NY, USA) pre-coated with Matrigel. Cells (2×105) were resuspended in serum-free medium and seeded into the upper chamber. Culture medium containing 20% fetal bovine serum (FBS) was added to the lower chamber as the chemoattractant. The cells were incubated in a humidified incubator at 37°C for 24 h (migration assay) or 36 h (invasion assay). Non-invading cells in the upper chambers were removed with cotton swabs. The cells attached to the lower surface were fixed and stained. The number of cells which attached to the lower surface was counted in five random fields under a microscope (×200).
9
Cell Migration and Invasion Assay
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A Transwell migration and Matrigel invasion assay was used to determine the migration and invasive ability of the A549 and NCI-H1975 cells. For migration, 100 μl FBS free medium were added onto the upper surface of the migration chambers (cat. no. 3422; Corning, Inc.) and incubated at 37°C for 30 min. Subsequently, the FBS-free medium was discarded and a total of 1×105 cells in 100 μl FBS-free medium was added onto the upper surface of the migration chambers. The lower chambers were filled with 30% FBS medium. For invasion assay, a total of 500 μl of the mixture was added onto the upper surface of the invasion chambers (cat. no. 354480; Corning, Inc.), while the lower chambers were filled with 30% FBS medium. Following incubation for 24 or 48 h at 37°C, the culture medium and the cells attached on the upper surface were removed. Cells attached on the lower surface were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with Giemsa stain (cat. no. 32884; Sigma-Aldrich; Merck KGaA) for 30 min at room temperature. The images of migratory and invasive cells were collected using a light microscope (Olympus Corporation; magnification, ×100).
10
Transwell Assay for Cell Migration and Invasion
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Transwell inserts were coated with a 2% gelatin solution and incubated at RT for 4 h for the migration assay. The gelatin-coated transwell inserts (353097, Falcon BD, Bedford, MA) and invasion chambers (354480, Corning, NY) were rehydrated in serum-free medium. Complete medium with 20% FBS (700 μl) served as a chemoattractant in the bottom chamber, and 5 × 104 cells/well were incubated for 24 h at 37°C with 5% CO2. At the end of the incubation period, migrated and invasive cells were fixed with methanol for 5 min and stained with 0.1% crystal violet.
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