Cell Seeding and Invasion Through a Collagen I Matrix
- Oris™ exclusion zone technology for unambiguous analysis
- Real-time and end-point measurements
- No artificial membranes for cells to pass through
- Readout with microscopes, plate readers, and high-content instruments
In the 3D Embedded Invasion Assay, cells are suspended in a thick layer of collagen I surrounding a 2 mm circular central cell-free collagen I detection zone (left image). After several days incubation, invading cells move into the detection zone (right image) where they can be unambiguously quantified. Images are false-color composites of the detection zone stained for nuclei (red) and actin (green).
Cell Movement in the Exclusion Zone is Specifically Inhibited by MMP Inhibitor GM6001
Matrix metalloproteinase (MMP) inhibitor GM6001 blocks movement of HT1080 cells through the collagen matrix in the Oris™ 3D Embedded Invasion Assay but not cell movement in the Oris™ Cell Migration Assay, which monitors 2D migration across the plate surface.
30,000 cells were seeded per well in Oris™ 3D Embedded Invasion Assay plates (3D, blue) or Oris™ Cell Migration Assay (2D, red) plates. Culture medium ± drug was added to wells after collagen in the exclusion zone was allowed to polymerize (3D) or after stoppers were removed following cell adhesion (2D). After 1 day (2D) or 5 days (3D) cells were fixed and stained with DAPI (3D) or TRITC-Phalloidin (2D), then imaged on a Zeiss Axio Observer.Z1 microscope with automated stage. Images were analyzed using ImageJ. 3D invasion was quantified by counting cells in each of six focal planes at 0-500 µm above the plate bottom in 100 µm increments, summing cells across all planes for each well. 2D migration was quantified by measuring percent area closure in the exclusion zone. Data points are average of six wells ± standard deviation.
Assays results can be quantified with plater readers or with image analysis
30,000 HT1080 cells were seeded per well in the Oris™ 3D Embedded Invasion Assay. Culture medium ± GM6001 was added after collagen in the exclusion zone polymerized. After 7 days incubation, cells were fixed and stained with DAPI. 3D invasion was quantified (i) by image analysis of cells invading into the exclusion zone (blue data points) and (ii) using the mask and a plate reader to measure fluorescence intensity in the exclusion zone (green data points). All data points average of six wells ± sd.
3D Cell Distribution
After 5 days incubation in the Oris™ 3D Embedded Invasion Assay, cells were stained with DAPI, then images of each well were captured in 7 focal planes 0-600 µm above the plate bottom. Cells were counted using ImageJ to define the detection zone and to count in-focus cells using a brightness threshold, (four wells per data point). The images to the right of the histogram are ImageJ reconstructions of in-focus cells at each height. Cells are distributed throughout the height of the collagen I matrix.
|Product Number||Product Description||Price|
|EIA1||Oris™ 3D Embedded Invasion Assay, 1-packOris™ Collagen I Coated, 96-well plate with Oris™ Cell Seeding Stoppers, 1Oris™ Detection Mask, 1Oris™ Stopper Tool, 1Oris™ Collagen I (Rat tail) 5 mg/mL, 2 x 2 mL||$355|
|EIA3||Oris™ 3D Embedded Invasion Assay, 3-packOris™ Collagen I Coated, 96-well plate with Oris™ Cell Seeding Stoppers, 3Oris™ Detection Mask, 1Oris™ Stopper Tool, 1Oris™ Collagen I (Rat tail) 5 mg/mL, 6 x 2 mL||$995|
|EIAST||Oris™ 3D Embedded Invasion Assay, starter pack, for 48 wellsOris™-compatible 96-well Collagen I Coated plate, 1Oris™ Cell Seeding Stoppers, 2 x 24Oris™ Detection Mask, 1Oris™ Stopper Tool, 1Oris™ Collagen I (Rat tail) 5mg/mL, 1 x 2mL||$255|
|CMA.IRT||Oris™ Stopper Tool||$25|
|CMA.MM||Oris™ Detection Mask, pkg 5||$45|
Oris™ is a trademark of Platypus Technologies, LLC. Oris™ technology is protected by US patents 7,842,499, 8,268,614 and 8,512,974 and pending applications.