The in vitro cell invasion assay was performed using BD BioCoat Cell culture inserts (8.0μm, 24-well format, BD Biosciences, Cat#354578) following the manufacturer's protocol with the following modifications. The cell culture inserts were evenly coated with diluted Matrigel (0.5 mg/ml dilution with serum free DMEM medium) and incubated at 37°C for 4 hours. During the last hour of incubation, the lower chamber was filled with 700 μl DMEM medium containing 10% horse serum. In triplicate, 2×105 PaTu8988T cells in DMEM medium with 0.1% BSA were added to the upper chamber with the addition of 100U of rMMP3 and incubated at 37°C for 18 hours. Cells on the top side of the membrane were removed with cotton-tipped swabs, and the inserts were fixed with 100% methanol at -20°C for 20 minutes, and stained with 0.5% crystal violet for 1 hour. Pictures of the stained inserts were taken using a 2× objective lens and counted using Image-Pro 6.3 software (Media Cybernetics). The results are presented as the relative invasion compared to untreated control cells.
Biocoat cell culture insert
Manufactured by BD
Sourced in United States
BioCoat cell culture inserts are a laboratory equipment product designed to facilitate in vitro cell culture experiments. The inserts provide a permeable membrane support for cell growth and migration studies.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel basement membrane matrix, by BD (4 mentions)
Matrigel, by BD (3 mentions)
Collagen type 4, by BD (2 mentions)
Diff quick staining kit, by Siemens (2 mentions)
Image pro 6, by Media Cybernetics (1 mentions)
Uplan fl 10x 0.30, by Olympus (1 mentions)
Sema3e, by R&D Systems (1 mentions)
Osteomeasure system, by OsteoMetrics (1 mentions)
Cytoselect cell adhesion assay kit, by Cell Biolabs (1 mentions)
Differential quik stain kit, by Electron Microscopy Sciences (1 mentions)
Matrigel basement membrane matrix, by Corning (1 mentions)
Laminin, by Merck Group (1 mentions)
Collagen type 4, by Merck Group (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Biocoat matrigel invasion chamber, by BD (1 mentions)
21 protocols using biocoat cell culture insert
1
In Vitro Cell Invasion Assay
2
In Vitro Cell Invasion Assay
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In vitro invasion assay was performed using BD BioCoat Cell Culture Inserts coated with Type IV Collagen (BD Biosciences, Bedford, MA, USA) as described previously (Kurihara et al, 2013 (link)). In brief, cells were suspended in 500 μl of DMEM and placed in the insert. After incubation for 48 h at 37 °C, the filters were dyed with a Diff-Quick Staining Kit (Siemens Healthcare Diagnostics, Newark, DE, USA). The stained cells were counted in whole inserts at × 100 magnification. Each experiment was repeated at least three times.
3
Cell Migration Assay with Collagen
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A modified Boyden chamber assay was performed using BD BioCoat Cell Culture Inserts coated with Type IV Collagen (BD Biosciences, Bedford, MA, USA). Briefly, cells were suspended in 500 μL of DMEM and placed in the insert. After incubation for 48 h at 37 °C, the filters were dyed with a Diff-Quick Staining Kit (Siemens Healthcare Diagnostics, Newark, DE, USA). The stained cells were counted in whole inserts at 100× magnification. Each experiment was repeated at least three times.
4
Transwell Migration Assay for Breast Cancer
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For transwell migration assays (BD Biosciences, BIOCOAT® Cell culture inserts, 354578), 2 × 104 breast cancer cells per well were allowed to migrate through 8-μm pores towards control medium (α-MEM + 1% FBS + 1% P/S, referred to as Ctrl), ObCM, or towards α-MEM supplemented with recombinant Semaphorin 3E (Sema3E, R&D Systems, 100-500 ng/ml) for 6 h. Migrated cells were stained using Giemsa’s azur eosin methylene blue solution (Merck, HX8389304). Cells within 4 fields of view of interest were counted using the OsteoMeasure system (Osteometrics) using a × 10 objective (Olympus UPlan Fl 10x/0.30 ∞/).
5
Transwell Invasion Assay Protocol
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Invasion was measured using 24-well BioCoat cell culture inserts (BD Biosciences) with an 8-μm-porosity polyethylene terephthalate membrane coated with Matrigel Basement Membrane Matrix (BD Biosciences). The invasion assay was performed as previously described (35 (link)).
6
Cell Invasion Assay with Matrigel
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Invasion was measured using 24-well BioCoat cell culture inserts (BD Biosciences, NJ, USA) with an 8μm porosity polyethylene terephthalate membrane coated with Matrigel Basement Membrane Matrix. Normal, ZNF703 inhibited and overexpressed cells in serum-free medium (2×105 cells/200 μl) were added to the upper chambers while the bottom chambers containing 10% FBS were utilized to supply a chemoattractant. Cells were permitted to migrate through the porous membrane for 24 h at 37°C with 5% CO2 while non-migrating cells remained on the upper surface of the filter. Cotton swabs were utilized to gently remove those non-migrating cells before fixing the migrated cells on lower surface with 100% methanol. Migrated cells were then stained by crystal violet and counted under a fluorescent microscope (Nikon Model Eclipse Ni-E, x100). Each experiment was independently performed three times.
7
FTY720 Invasion Assay
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Eight hours after FTY720 treatment, invasion was measured using 24-well BioCoat cell culture inserts (BD Biosciences, NJ, USA) with an 8 μm porosity polyethylene terephthalate membrane coated with Matrigel Basement Membrane Matrix.
8
Cell Invasion Assay using Matrigel
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A cell invasion assay was performed in BioCoat cell culture inserts with a polystyrene membrane (8-μm pore; BD Bioscience) in a 24-well tissue culture plate. The culture insert was coated with Matrigel (BD Bioscience, 8.7 μg per chamber); the lower chamber was filled with DMEM containing 10% serum. A total of 4 × 105 MCF7 cells or 1 × 105 SK-BR-3 cells were seeded in the upper chamber containing DMEM with 10% serum and the cells were incubated at 37°C for 24 h. After wiping off the cells on the upper side, the membrane was removed and stained with Giemsa solution. The cells that had migrated to the lower side of the membrane were counted under a microscope.
9
Quantifying Cell Invasion through Matrigel Transwell Assay
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Invasion was measured using 24-well BioCoat cell culture inserts (BD Biosciences) with an 8-μm-porosity polyethylene terephthalate membrane coated with Matrigel Basement Membrane Matrix (100 μg/cm2). Briefly, the Matrigel was allowed to rehydrate for 2 h at 37C by adding warm, serum-free DMEM. The wells of the lower chamber were filled with medium containing 5% fetal bovine serum. U87 and LN229 Cells (5 × 104) were seeded in the upper compartment (6.25-mm membrane size) in serum-free medium. Exogenous CCL27 (0.1μg/ml) were added in the upper chamber. After 24h incubation, non-invading cells were removed from the top well with a cotton swab, and bottom cells were fixed with 3% paraformaldehyde, stained with 0.1% crystal violet, and photographed in 9 independent 10× fields for 3 wells. Fold invasion was calculated relative to blank control. Data represents mean ± standard error (SE) of 3 independent experiments.
10
Cell Invasion Assay Using Matrigel
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Invasion was measured using 24-well BioCoat cell culture inserts (BD Biosciences) with an 8-μm-porosity polyethylene terephthalate membrane coated with Matrigel Basement Membrane Matrix (BD Biosciences). The invasion assay was performed as previously described [25 (link)].
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