FAQ's for the Oris™ Cell Migration Assembly Kits in designing cell migration, cell invasion, and 2-D closure (wound) assays.
How do I know if I have properly inserted the Oris™ Cell Seeding Stopper into each microplate well?
The tip of a properly inserted stopper will create a bullseye pattern at the bottom of the well. To view this bullseye pattern, turn the plate over after inserting the stoppers and tilt the plate at an angle. You will be able to see this bullseye 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. Click here for a view of the bullseye pattern.
How reproducible are the cell migration, cell invasion and 2-D closure assays using the Oris™ Assembly kits?
Results obtained using the Oris™ Assembly kits show slightly higher Coefficients of Variations (CV) values for these assays since the Oris™ Cell Seeding Stoppers are inserted by hand as opposed to the Oris™ kits where the stoppers are pre-inserted by automation. Quality Control testing has indicated that CV's are dependent upon the cell line studied, length of time for incubation, and staining techniques. Platypus has observed CV values from 12%-20%.
What is the stability of the Oris™ Assembly Kits?
These kits are stable at 4°C for 12 months.
What cell lines can be used with the Oris™ Assembly Kits?
Any cell line that is adherent can be used to design migration, invasion, or 2D closure (an alternative to wound healing or scratch assay) assays. The following are a few examples::
What types of cell-based studies can the Oris™ Cell Migration Assembly Kits be used to perform?
The Oris™ Cell Migration Assembly Kits can be used to design chemokinesis assays, perform morphological analysis, detect cell invasion, 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 pins on the bottom of the 96-well plate. Finally, gently press the mask into place ensuring that the mask is snapped 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 plates supplied in the Oris™ Cell Migration Assay kit, the Oris™ Cell Invasion Assay kit, or the Oris™ Cell Migration Assembly kits.
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 migration 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™ Assays?
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, the stoppers have been sterilized and Platypus can not guarantee the integrity of the stoppers after a second sterilization process since 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 at an angle, 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 Assembly Kit?
No, the Oris™ Cell Migration Assembly kits have 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) or a digital imaging instrument.
If I am using fluorometric readout of the results of my cell migration, invasion or 2-D closure 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 these cell motility assays, 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, invasion or 2-D closure.
Are there dyes that are incompatible with the Oris™ Cell-based Assays?
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. Examples are ........
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 not to 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™ Assay compatible with top and bottom reading fluorescence plate readers?
The Oris™ Assay (migration, invasion or 2-D closure) is compatible with bottom probe plate readers. Examples of compatible machines include:
Will the entire Oris™ 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.
How quantitative is an Oris™ 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, fluorophor 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
