FAQ's for the Oris™ line of Assays (Oris™ Cell Migration Assay, the Oris™ Cell Migration Assay - Collagen I Coated, and the Oris™ Cell Migration Assay - Fibronectin Coated)
How reproducible is the Oris™ Cell Migration Assay?
Results obtained using the Oris™ Cell Migration Assay are highly reproducible when compared to current assays. Quality control testing has indicated Coefficients of Variances that range from 9-12%.
What is the stability of the Oris™ Cell Migration Assay?
The Oris™ Cell Migration Assay is stable at 4°C for 6 months.
How do I store the Oris™ Cell Migration Assay?
The Oris™ Cell Migration Assay should be refrigerated at 4°C.
What cell lines can be used with the Oris™ Cell Migration Assay?
Any cell line that is adherent can be used with the Oris™ Cell Migration Assay. The following are a few examples:
What cell lines can be used with the Oris™ Cell Migration Assay - Collagen I Coated and the Oris™ Cell Migration Assay - Fibronectin Coated kits?
Any cell line that is adherent can be used with the Oris™ Cell Migration Assay - Collagen I Coated kit and the Oris™ Cell Migration Assay - Fibronectin Coated kit. The following are a few examples:
What types of cell migration can the Oris™ Cell Migration Assay be used to study?
The Oris™ Cell Migration Assay can be utilized to detect chemokinesis, perform morphological analysis, and mimic a 2-D closure/wound assay.
How is the Oris™ Detection Mask attached to the 96-well plate?
Prior to adding any liquids to the wells of your 96-well plate, familiarize yourself with the attachment and removal of the Detection Mask. To do so, orient the chamfered corners of the mask with the chamfered corners of the 96-well plate, ensuring that the A1 corner of the mask is aligned with the A1 well of the plate. Then, align the holes in the mask attachment lugs with the bosses on the bottom of the 96-well plate. Finally, gently press the mask into place ensuring that the mask is flat against the bottom of the 96-well plate.
Can I attach the Detection Mask to any type of 96-well plate?
No, due to the engineering specifications of the detection mask, it will only attach to the plate supplied in the Oris™ Cell Migration Assay kit.
Do I have to apply the Detection Mask at the beginning of an assay?
No, the mask may be applied at any point during the assay. For kinetic assays, it is often most convenient to apply the mask at the beginning of the assay before any liquids are placed in the well. For endpoint assays, using fixed and stained cells, it is often most convenient to apply the mask just before reading.
What if I forgot to attach the Detection Mask before I added my samples to the wells?
Once you have familiarized yourself with the technique of attaching and removing the detection mask, you may apply it after liquids have been placed in the wells.
Are there any hints for adding my cells to the wells through the side ports of the Cell Seeding Stoppers?
Add or extract cells and media by placing the pipette tip along the wall of the well. Care should be taken not to disturb the Cell Seeding Stopper when introducing the pipette tip into the well. A gel loading tip may be useful.
In some of the wells, the contents were not uniformly distributed. Is there something that I can do to address this issue in the future?
Yes, lightly tap the plate on your work surface to evenly distribute well contents.
I typically aspirate media from the wells using a vacuum. Will this method work with the Oris™ Cell Migration Assay?
No, due to the potential to dislodge the cell layer, we urge you not to aspirate media from the wells using a vacuum. Care should be taken throughout all steps of the assay not to disturb the cell layer.
Are the Oris™ Cell Seeding Stoppers reusable with new sterile plates?
No, due to the specificity with which the stoppers centered in each well of a 96-well plate and due to engineering specifications, the stoppers are not reusable. In addition, the stoppers cannot be autoclaved (repeated autoclaving will cause damage).
Can the Oris™ Cell Seeding Stoppers be cleansed with ethanol or isoproponal?
No, alcohol cleansing may not effectively sterilize the stoppers due to the nature of the stopper material.
Should the Oris™ Cell Seeding Stoppers be removed in order to add a substrate to the bottom of the plates and then reinserted?
No, as mentioned above, due to the specificity with which the stoppers are centered in each well of a 96-well plate and due to engineering specifications, the stoppers should not be removed and reinserted.
Can I use only a few of the wells and utilize the rest for another experiment(s) at a later date?
Yes, the Oris™ Cell Seeding Stoppers are grouped into strips comprised of 4 stopper tips. This allows the user to remove any factor of 4 stoppers at a time and leave the remainder of the stoppers available for future experiments. Note that care must be taken to maintain the sterility of the plate.
Are there any hints for using the Stopper Tool to remove the stoppers?
Yes, most importantly, do not use the stopper tool as a lever to pry the stoppers from the well, as doing so may cause displacement of the seeded cells. To remove the stoppers, first secure the 96-well plate by holding it firmly against the deck of your work space. Next, slide the tines of the stopper tool between the top of the stopper strip backbone, keeping the underside of the stopper tool flush with the top surface of the plate. Finally, lift the stopper tool vertically to gently remove the stopper.
Do I have to use a fluorescent stain and a fluorescence plate reader to obtain my results with the Oris™ Cell Migration Assay?
No, the Oris™ Cell Migration Assay has been designed to be used with any commercially available stain or labeling technique and the readout can be performed with a plate reader or by microscopic examination (i.e., cell counting or image capture / analysis).
If I am using fluorometric readout of the results of the Oris™ Cell Migration Assay, what type of a fluorescent dye would you recommend I use to label cells?
Fluorescent dyes derived from the fluorescein, rhodamine and cyanine families can all be used with this system, so long as their emission wavelengths are within 490-700 nm. Note: the fluorescence stain used must uniformly stain the cells. The utilization of a fluorescence probe that is affected by experimental conditions will increase the variability of results and reduce the correlation between fluorescence signal and cell migration. Fluorescence probes that are affected by experimental conditions could be utilized as a counter-stain for the study of factors and processes that affect cell migration.
Are there dyes that are incompatible with the Oris™ Cell Migration Assay?
No, however, the fluorescence stain used must uniformly stain the cells.
Can cells be labeled with more than one dye?
Yes, as long as the emission wavelengths of both dyes fall between 490-700 nm (and the emission wavelength of the first dye does not overlap with the excitation wavelength of the second dye), and your detection equipment has appropriate filters to distinguish the dyes, multiplex detection is possible.
My cells take several days to migrate. Will I be able to detect my cells with fluorescence after 2 days?
For slowly migrating cells, we suggest choosing a stable dye that will still be functional after several days.
I have noticed that my background reading seems extremely high and upon further evaluation, the detection mask and 96-well plate seem to be contributing to the fluorescence. Is there something that I can do to address this issue?
Please take care that you do not contaminate the bottom of the 96-well plate and the detection mask with any substance that may autofluoresce. To remove dust and any other contaminants, wash the detection mask and the bottom of the 96-well plate with 70% ethanol and dry off.
Is the Oris™ Cell Migration Assay compatible with top and bottom reading fluorescence plate readers?
The Oris™ Cell Migration Assay is compatible with bottom probe plate readers. Examples of compatible machines include:
Will the entire plate with lid fit in my plate reader?
Some plate readers have limited clearance and may not accept the plate routinely with the lid on. If you experience clearance problems, try removing the lid prior to measurement.
Can the Oris™ Cell Migration Assay be used to quantify the number of migrating cells?
Yes, as long as cell proliferation is taken into account. You will then be able to microscopically observe the cells or generate a standard curve and back-calculate the cell number.
How quantitative is the Oris™ Cell Migration Assay? Can I distinguish 100 cells from 200 cells?
Fluorescence techniques in general show sensitivity over a wide dynamic range. Your ultimate sensitivity and discrimination ability will be dependent upon your choice of cell type, fluorochrome, and detection equipment. In experimentation, we have been able to detect a change in as little as 50 cells.
Oris™ is a trademark of Platypus Technologies, LLC