A transwell assay was used to assess the invasion abilities of the GC cells. The cells (2×105 cells/well) cultured in a serum-free medium were seeded into the Matrigel-precoated (BD Biosciences, San Jose, CA, USA) upper chambers. A medium (600 μL) containing 100 ng/mL of chemokine stromal cell-derived factor-1 (Sino Biological, Beijing, China) was added to the lower chambers. After 24 h of culturing, the invasion cells were fixed in methanol and stained with 0.1% crystal violet for 30 min. Cells from 4 random fields were photographed and counted.
Matrigel precoated
Manufactured by BD
Sourced in United States
Matrigel-precoated is a basement membrane extracellular matrix preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma. It is a complex mixture of extracellular matrix proteins, including laminin, collagen IV, heparan sulfate proteoglycans, and entactin.
Automatically generated - may contain errors
Lab products found in correlation
Transwell chamber, by Corning (3 mentions)
Transwell insert, by Corning (2 mentions)
Complete dmem, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Optical microscope, by Olympus (1 mentions)
Transwell plate, by BD (1 mentions)
Inverted microscope, by Olympus (1 mentions)
24 well transwell plate, by Corning (1 mentions)
8 protocols using matrigel precoated
1
Transwell Invasion Assay for Gastric Cancer
2
Transwell-Based Cell Invasion Assay
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The Transwell chambers (Corning, Corning, NY, USA) used for invasion assay were Matrigel pre-coated (BD Biosciences, Franklin Lakes, NJ, USA). After suspending cells in serum-free medium at a cell density of 1.0 × 105/mL, the Transwell chambers were placed in a 24-well plate. The apical and basolateral chambers contained 200 μL suspension and 500 μL medium containing 10% FBS, respectively. Forty-eight hours later, the chambers were removed, and the cells were fixed using 5% paraformaldehyde for 20 min. The fixed cells were stained using 0.1% crystal violet. The invading cells in five randomly selected fields for each filter were enumerated using a light microscope (Olympus, Tokyo, Japan).
3
Matrigel-Based Transwell Invasion Assay
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Transwell invasion assays were performed using 8-µm pore Matrigel®-precoated (37°C for 3 h and room temperature overnight) polycarbonate membranes (BD Biosciences). Briefly, 3×104 cells were seeded into the upper chambers of Transwell plates in serum-free DMEM (Gibco; Thermo Fisher Scientific, Inc.). The lower chambers contained complete DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS. Following incubation at 37°C for 48 h, cells in the upper chamber were removed and the invasive cells on the lower surface of the membranes were fixed in 4% paraformaldehyde at room temperature for 15 min, stained with 0.5% crystal violet at room temperature for 20 min and visualized using a light microscope (magnification, ×100) (Olympus Corporation). ImageJ software (version 1.8.0; National Institutes of Health) was used for data analysis.
4
Cell Invasion Assay Protocol
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Indicated cells were re-suspended in serum-free RPMI medium to a concentration of 1 × 105/mL, and 100 μL were added into the upper chamber of matrigel-precoated (BD BioSciences, CA, USA) transwell inserts (Corning, NY, USA). The lower compartment was supplemented with complete RPMI medium. After 12 h of incubation, cells free in the upper chamber were wiped off with cotton swab. The invaded cells were fixed and stained with 0.25% crystal violet, the typical images were captured and invaded cells were counted in five random fields under the microscope.
5
Cell Migration and Invasion Assay
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2*10^4 cells were seeded into a 24-well transwell chamber (8mm, Corning, US) for migration assay or into a Matrigel-precoated (BD Biosciences) chamber for invasion assay. The lower chamber was filled with 0.5mL RPMI 1640 medium supplemented with 10% FBS. After incubation at 37°C for 48h, cells in the upper chamber were removed. Cells on the surface of the lower chamber were fixed with 4% formaldehyde for 15min and were stained with 0.5% crystal violet for 20min. The results were observed using a microscope (X200, Olympus, JP). ImageJ software (NIH, US, version 1.6.2) was used for data analysis.
6
Transwell Cell Invasion Assay
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The invasion assay was performed using transwell chambers (Corning, New York, USA) with 50 μL Matrigel-precoated (BD, San Diego, USA) polycarbonate membrane (8.0 μm pore size) as described in the previous report [35 (link)]. Briefly, cells were collected and resuspended in the serum-free RPMI 1640 medium at a concentration of 1 × 105 cells/mL, respectively. Then, the cell suspensions were added into the top chambers (200 μL/well) and the bottom chambers were filled with RPMI 1640 medium containing 10% FBS (600 μL/well), followed by a 24 h incubation at 37°C. The cells that did not penetrate the polycarbonate membrane were swabbed using cotton bud; then, the cells that transmembraned through and adhered to the bottom of polycarbonate membrane were stained with 4′,6-diamidino-2-phenylindole dye (1 μg/mL) for 10 min, photographed under an Olympus fluorescence microscope, and counted manually. The average of three randomly selected ×400 fields' cell counts was recorded as the value of each chamber. The experiment was repeated twice with triplicate measurements in each experiment.
7
Cell Migration and Invasion Assay
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Cell migration assay was performed in a 24-well plate using Transwell inserts (Corning Inc., Corning, NY, USA) with 8.0-µm pore size. The primary spheres and spheres at 4 passage were collected and resuspended in DMEM and CSC-M, respectively. Then, 200 µL of growth factor-free CSC-M containing 5 × 104 cells was loaded onto the upper chamber of the transwell insert, and 600 µL of CSC-M was loaded onto the lower chamber. Cells were allowed to migrate for 24 h at 37 °C. The inserts were washed with PBS, and non-migratory cells that remained in the upper compartment were removed with a cotton swab. The migratory cells in the lower chamber were fixed with methanol and stained with crystal violet (Hengxing Chemical Reagent Co., Ltd., Tianjin, China), and counted using an optical microscope (magnification, x400; Olympus, Tokyo, Japan). Cell invasion assay was performed using matrigel-precoated (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) transwell chambers (8-µm pore size; Corning Inc., Corning, NY, USA) in a 24-well plate.
8
Transwell Assay for Cell Migration and Invasion
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Migration and invasion experiments were performed with PC cells using a 24-well Transwell plate (Corning). For the migration assay, stably transfected cells (PANC-1) in 100 µl of serum-free DMEM were plated in the upper chamber. Then, 600 µl of DMEM supplemented with 30% FBS was placed in the lower chamber. After the cells were incubated at 37°C for 24 h, the cells that could not migrate were removed from the upper surface of the membrane with a cotton swab. The cells that had passed through the membranes were stained with 0.1% rystal violet for 10 min. The migrating cells were viewed under an inverted microscope (Olympus, Tokyo, Japan), and nine random fields (magnification, ×200) were photographed and counted.
For the invasion assay, stably transfected cells (PANC-1) in 500 µl of serum-free DMEM were plated in the upper chamber of Matrigel-precoated (Becton-Dickinson Co., NJ, USA) Transwell plates. Then, 750 µl of DMEM supplemented with 30% FBS was placed in the lower chamber. After the cells were incubated at 37°C for 40 h, the non-invading cells were removed from the upper surface of the membrane, and the cells on the lower surface of the insert were stained and counted as the migration assay. The experiment was performed three times.
For the invasion assay, stably transfected cells (PANC-1) in 500 µl of serum-free DMEM were plated in the upper chamber of Matrigel-precoated (Becton-Dickinson Co., NJ, USA) Transwell plates. Then, 750 µl of DMEM supplemented with 30% FBS was placed in the lower chamber. After the cells were incubated at 37°C for 40 h, the non-invading cells were removed from the upper surface of the membrane, and the cells on the lower surface of the insert were stained and counted as the migration assay. The experiment was performed three times.
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