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Millicell® μ-Migration Assay Kit

Stable gradient for quantitative, real-time, slide-based assays

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What equipment and software do I need to track migrating cells?You will need a phase contrast microscope or fluorescence inverted microscope, heating stage, time-lapse video equipment (CCD camera, video camera, acquisition software), motorized stage and autofocus (x, y, z) and the NIH Image J plug-in software. Instructions for free download of this software are available at www.millipore.com/umigration).
Can I conduct the migration assaywith fluorescently stained cells?Yes. Fluorescent staining is compatible with the migration kit. The cells are pre-stained before loading into the slide.
Can I use a normal digital camera connected to a microscope to take images manually at different time points (e.g. if the microscope doesn’t have video equipment)? If so, can this data be opened in the Image J plug-in software and analyzed?Yes. To analyze the pictures with the Image J software, just follow these steps: 1. If taking pictures manually at different time points, be sure to take them with the slide always at the same position on the microscope. 2. Save the image files in order. 3. Import the files into Image J—the software will organize the images in order. 4. Analyze the data using Image J software.
Can I take images of assays running concurrently in different chambers on the same slide?This is easily done with a microscope that has a computer-controlled movable stage. If the microscope doesn’t have a computer-controlled movable stage, it is very difficult to manually take all the pictures/videos from 3 chambers at different time points (or at the same time) since the registration (original point) is lost for each of the different chambers. The migration of cells is very small, usually several microns/hour. If the photos are taken manually in a slightly different area, the trace of the cells is not accurate. In this case, we recommend running one chamber at a time in individual assays.
If multiple assays are run on a slide and images are captured for all of them, can the Image J software identify which chamber each photo derives from at different time points?No, Image J software cannot sort the photos taken of simultaneous assays. You will need to sort the pictures before loading into Image J.
How can I correctly track the recommended 20 to 50 cells which are necessary to obtain representative data in one image/frame?Please refer to the Image J plug-in software document available at www.millipore.com/umigration.
What the effective surface area of the viewing area?The effective viewing area depends on the objective magnitude of the microscope used. The appropriate magnitude should cover the entire area encompassing the cells, the source, and the paths of the cells migrating towards/against the source (1 mm, y-direction).
I am using fibronectin and I never let it dry out before seeding cells. However, the protocol suggests drying the slide out completely. Can I seed the cells immediately after coating like I normally do when culturing my cells?No. As this is a microfluidic device, it is critical to ensure complete evaporation of fluid from the slide prior to cell loading so that air bubbles do not get trapped in the channels.
Do I need to worry about CO2 or O2 gas exchange?For CO2 dependent cell lines, CO2 gas exchange is necessary during real-time imaging. The slide’s plastic is gas-permeable thus allowing for some gas exchange to occur. A CO2 gas exchanger is optimal.
Can the conditioning or incubation of the chambers be shortened at all?No. As this is a microfluidic device, it is critical to ensure complete evaporation of all fluid from the slide prior to cell loading so that air bubbles do not get trapped in the channels.
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