(96-well w/ Stoppers)
|Oris™ Pro Plates
(96-well w/ BCG)
|Oris™ Pro 384 Plates
(384-well w/ BCG)
|Diameter of Well – Bottom||6.3 mm||6.58 mm||3.3 mm|
|Diameter of Well – Top||6.45 mm||6.96 mm||3.7 mm|
|Diameter of Cell Free Detection Zone||2 mm||2 mm||2 mm|
|Well Volume||400 µL||392 μL||131 µL|
|Suggested culture medium per Well||100 µL||100 µL||20 µL|
|Area of Outer Annular Region (cell seeding region)||28.03 mm2||30.86 mm2||6.86 mm2|
|Area of Central Detection Zone||3.14 mm2||3.14 mm2||3.14 mm2|
|Plate Height||14.85 mm||14.4 mm||14.4 mm|
|Plate Height with Lid||17.9 mm||17 mm||16.5 mm|
|Offset of Wells (A-1 location, X)||14.32 mm||14.38 mm||12.13 mm|
|Offset of Wells (A-1 location, Y)||11.25 mm||11.24 mm||8.99 mm|
|Distance between Wells||9.0 mm||9.0 mm||4.5 mm|
|Well Depth||12.1 mm||10.9 mm||11.5 mm|
|Thickness of Well Bottom||0.25 mm||190 μm ±10%||190 μm ±10%|
|Storage||4°C||15 – 30°C||15 – 30°C|
If your imaging system is provided with a software package, your vendor can help you with Oris™ quantification, and may already have a routine specifically designed for Oris™. See this application note for an example.
Some plate readers have limited clearance and may not accept the plate with the lid on.
The mask provided with the Oris™ assays has an aperture diameter of 2.1 mm while the size of the Detection Zone formed by the stopper is 2.0 mm. The larger mask aperture is designed to allow some fluorescent background signal to included in the light path in order to bring the detector into the appropriate dynamic range, as illustrated in this technical note.
Cells are typically visible under bright field optics without staining, especially if phase contrast optics are available. However, staining makes cells easier to see, and can reveal physiological states that bright field optics cannot. The Oris™ and Oris™ Pro Assays impose no restrictions on choice of stain, and you can use multiple stains simultaneously if desired.
To quantify migration/invasion via area closure, we recommend a fluorescent cytoplasmic stain such as TRITC-phalloidin in order to maximize signals for your detector.
To quantify migration/invasion by counting cells, we recommend a nuclear stain such as DAPI. Restricting the stain to the nuclei creates smaller objects for imaging than do cytoplasmic stains, providing greater separation between individual cells and therefore more accurate counts.
You may also consider pre-labelling cells prior to seeding in the Oris™ assays. However, some stains may impact migration and/or invasion, potentially creating experimental artefacts.
Oris™ and Oris™ Pro assays are designed to work with any plate reader, microscope, or high content imager that is compatible with the industry-standard microplate format. Several application notes detail the use of Oris™ with plate readers and high content imagers. The table below summarizes your options:
|Instrument||Oris™||Oris™ Pro & Pro 384||Comments|
|Standard Plate Reader||X||Requires fluorescent staining and Oris™ physical mask. Plate reader should have the detector below the plate|
|Plate Reader with Virtual Masking Software||X||X||Requires fluorescent staining. Check with vendor that software can mask the 2 mm central circular detection zone|
|Microscope, Inverted||X||X||Automated motor-driven stage and camera recommended for efficient analysis|
|High Content Imager||X||X|
|Platypus Bill||The Platypus bill can sense many things but alas can’t quantify Oris™ assays|
No. Most vacuums are too powerful, and will aspirate cells as well as medium. Instead, use a pipet or an automated liquid handling device to remove culture medium from assay wells.
There are several options, simplest first:
- Run the assay for less time than the cell doubling time to limit proliferation.
- Add an inhibitor of proliferation such as mitomycin C or actinomycin D that does not inhibit migration or invasion
- Immunostain with an anti-Ki67 antibody, a marker found only on proliferating cells, and discount Ki67-positive cells from the results.
- Monitor the assay by video microscopy so each cell’s history during the assay. This a time-consuming and data-heavy approach requiring expensive equipment.
For Oris™ assays, add test compounds to the culture medium once the cells have attached and the stoppers have been removed, but before migration begins.
For Oris™ Pro Assays, add test compounds once the BCG has dissolved and the cells have attached, but before migration begins.
For both Oris™ and Oris™ Pro assays, adhesion time is dependent on cell line and plate coating, with attachment times. You may remove and replace the medium prior to adding test compounds in order to eliminate any non-adherent cells. In the invasion assays, compounds may be incorporated into the collagen I solutions and/or added to the culture medium above the collagen.
For Oris™ Stopper-Based Assays, lightly tap the plate on your work surface to distribute cells evenly after seeding the cells but before removing the stoppers.
The assay plate may have been jolted after removal of Oris™ Stoppers or dissolution of the Oris™ Pro gel. Be careful when transferring the plates from work surface to incubator.
The cell line may be poorly adhered to the plate surface. Possible solutions are:
- If you used a TC-treated plate, try a collagen I- or fibronectin-coated plate for better adhesion.
- If using Oris™ assays, allow a longer time for cells to adhere before adding culture medium.
- If using Oris™ Pro assays, try reducing the volume in which cells are seeded so they reach the plate surface more quickly.
- If cell density is high, try seeding fewer cells so that all cells have the opportunity to adhere.
Pre-migration/invasion reference wells are used to establish the size and position of the detection zone in order to quantify the extent of movement in experimental wells.
For Oris™ assays, you can leave some stoppers in place until you read the results. Since Oris™ Pro assays lack stoppers, this method is not applicable.
For Oris™ Pro and Oris™ assays, alternative methods to establish reference wells are:
Simply collect images at time zero as migration is about to begin.
Add fixative to replicate wells or a replicate plate at the beginning of the assay. However, when fixing cells in a subset of wells in the plate, this method risks impacting cells adjacent to fixed wells as they may be exposed to fixative vapors.
Add an inhibitor of migration or invasion, such as cytochalasin D, to reference wells at the beginning of the assay. Be sure to add sufficient inhibitor to block cell movement completely.
You may wish to read a related application note here.
First secure the plate by holding it firmly against your work surface. Next, slide the tines of the stopper tool between the top of the stopper strip backbone, keeping the underside of the stopper tool parallel with the top surface of the plate. Finally, lift the stopper tool vertically to remove the stopper gently. See a video of the technique here.
Do not use the stopper tool as a lever to pry the stoppers from the well, as doing so may cause displacement of the cells.
No. Although 96-well plates are “industry standard”, well dimensions vary between suppliers. Since the stopper must fit perfectly in order to generate the reproducible, tightly bounded exclusion zone, only Oris™ plates will work properly.
Each well in a 24-well plate is substantially larger than wells in 96-well plates. Consequently, each data point requires more cells, more culture medium, more additives, and more time to complete the assay. That’s expensive. Moreover, 24-well migration and invasion assay plates provided elsewhere cost about the same or more than Oris™ 96-well plates, so your costs quadruple per data point before you even start the assay.
The tip of a properly inserted stopper will create a bull’s-eye pattern at the bottom of the well. To view this bull’s-eye pattern, turn the plate over after inserting the stoppers and tilt the plate at an angle. You will be able to see this bull’s-eye pattern in the center of each well through the clear bottom surface. Stoppers that have not sealed well can be re-inserted until the tip is properly placed. See a video of the technique here.
No. The stoppers must be stored refrigerated until use to preserve the perfect structural fit that generates the reproducible, tightly bounded exclusion zone. Stoppers that have been held at 37°C for any length of time will not fit properly, leading to irregular, variable exclusion zones.
Most Oris™ and all Oris™ Pro kits are configured for use in a single experiment. For Oris™, the stoppers must be stored refrigerated until use to preserve the perfect structural fit that generates the reproducible, tightly bounded exclusion zone. For Oris™ Pro, the humidity in the incubator hydrates the gel in the unused wells, which will create variability in exclusion zone size and gel dissolution time in subsequent experiments.
If you need fewer than 96 wells per experiment, consider the Oris™ Cell Migration Assembly Kit – FLEX, which includes four 96-well plates and four packs of 24 stoppers. Use each plate only once, with any number of stoppers that is a multiple of four.
For the best statistics, choose an assay time that results in untreated cells closing at least 2/3 of the original open area of the detection zone, but less than 100% closure. An advantage of the Oris™ and Oris™ Pro Cell Migration and Invasion Assays is that you can inspect the experiment at any time during the incubation period to ascertain how far the cells have moved. The first time you run an Oris™ assay, simply observe the cells periodically under a microscope to assess the extent of migration/invasion, and stop the experiment when the appropriate degree of movement is observed.
Since rates of cell movement differ widely between different cell types, optimal incubation times will vary with varying cell types. Migration times can vary from 16 – 72 hours, while invasion times can vary from 1 – 6 days. For extended experiments, we recommend that you change culture medium above with fresh inhibitors every 48-72 hr.
For Oris™ migration, Oris™ Pro migration and Oris™ Pro invasion assays, aim for 95-100% confluent monolayers around the cell exclusion zone at the start of the experiment. Seeding at a higher density may result in cells in the Detection Zone at the beginning of the assay while seeding at a lower density will yield fewer migrating cells in the detection zone after migration. Typically, 20,000 – 75,000 cells are seeded per well in Oris™ and Oris™ Pro 96-well plates, and 2,500 – 10,000 per well in Oris™ Pro 384-well plates. For your first experiment, we recommend testing several different cell seeding densities as described in Appendix 1 in the assay protocol.
For Oris™ 3D Embedded Invasion assays, we recommend seeding at 30,000 – 50,000 cells per well. As many as 75,000 cells per well may be seeded for some cell lines, but many invasive cells lines express high levels of matrix metalloproteinases that can degrade the collagen to the point where it collapses or even liquefies at high cell densities. Adding fewer cells ameliorates this problem.