Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. In this video article, A method for tracking hallmark events of mitosis is shown. Chromosome movement is monitored on an individual cell basis using cell lines expressing fluorescently tagged proteins.
Transfection with irna is directed against mitotic proteins, results in cell cycle changes that can be visualized by time-lapse imaging and quantified by image analysis. Hi, I'm Douglas Mackey from Katie Oman's lab at the University of Utah's Huntsman Cancer Institute. Today we will show you a procedure for imaging live cells as they progress through mitosis following SIA transfection.
We use this procedure in our lab to study any effects that disruption of proteins of interest may have on the timing of mitotic progression. So let's get started. Prepare a four wall lab tech two chambered cover glass by adding 0.5 milliliters of fibronectin to each.
Well incubate for 10 to 15 minutes at room temperature. In the meantime, prepare irna and lip RNAI max complexes for reverse transfection. According to the manufacturer's instructions for each chamber, well use the amount recommended for one well of a 24 well dish.
After 15 minutes, remove the fibronectin from the wells of the chambered cover glass. Then add 100 microliters of irna complexes to each well aliquot 20, 000 cells in 500 microliters to each. Well place the cells in the incubator for 24 hours.
The next day replace the transfection mixture with one milliliter of regular cell culture medium, then incubate cells an additional 24 hours before image acquisition. The image acquisition setup consists of a customized cell incubator that fits on the stage of an inverted microscope, which is controlled by image acquisition software. To prepare for image acquisition, ensure that the incubator is pre-warned and equilibrated.
Next, open the lid of the incubator and place the cells with the chamber lid on in the adapter. Use a small amount of modeling clay to hold it in place. Close the lid and position the entire incubator on the microscope stage ensuring that the incubator is firmly in place.
Next, using metamorph, set up the experiment by selecting apps multidimensional acquisition in the menu bar of the software. This will bring up a window in which the imaging parameters can be selected. Specify where to save the data files.
Then select brightfield and m cherry wavelengths using the four TX dry phase contrast objective, focus on a field of cells using the software, adjust the auto-focus function for the Brightfield channel only. This will allow the software to auto-focus at each stage position before each acquisition. For the first wavelength, capture an image Using a 50 millisecond exposure, adjust the exposure time to the minimum required to see a good image.
It is essential to limit the amount of light exposure to ensure sustained cell viability. As a general rule, this can best be achieved by using a neutral density filter and a longer camera exposure time. Rather than by increasing the illumination strength.
Repeat this procedure for any additional wavelengths. Then set the exposure time for each channel. Next, designate a time lapse interval of 15 minutes for eight hours at 15 stage positions.
If a better time resolution is desired, shorten the time interval between acquisitions. Remember that the more stage positions are used, the longer it will take to acquire. At each time interval, select and mark an appropriate number of stage positions to adequately sample the cell population.
Metamor will record the positions and return to the designated location for each acquisition. After the time lapse wavelength exposure, time and stage position information have been designated. Select the preview button on the screen to confirm that the software is controlling the equipment appropriately.
After ensuring that the system is functioning, select the acquire button on the screen to begin acquisition. Observe the automation through at least one full round of acquisition to ensure that everything is working properly. Then sit back and allow the computer and microscope to do the work.
To review the data, select apps, review multidimensional data in the menu bar, select the file location view, then select a stage position. Click on load images to review the stack of images from that position. Once the images have loaded, use the mouse to advance through the data frame by frame.
Here movies are generated of mitotic cells using metamorph. To make a movie in Metamorph select stack make movie. In the menu bar, select which frames to use the speed and file type of the movie.
Then select save to make a montage. First, save the data as a dot STK file in metamorph. Then open image J Open the dot s stk file in image J and crop a region of interest by marking the region and selecting image crop in the menu bar.
Then select image stacks. Make montage specify which frames to include the size and shape of the montage. And whether between frame borders are desired.
Then hit okay. Save the montage as a dot tiff file. Finally calculate time in each stage of mitosis designating the frame in which the first sign of DNA condensation or cell rounding is evident as T equals zero prophase.
Prometaphase ends when the chromosomes are aligned at the equator, metaphase will then proceed until the DNA begins to segregate anaphase telophase. And finally, cytokinesis follow and the cells begin to flatten. Time lapse imaging of hilar cells expressing his stone.
H two BCFP is shown. These cells were transfected with control irna and progress normally through the cell cycle. In comparison cells that were transfected with siRNAs directed against nuclear poring NUP 1 53 displace severe alterations in MIT progression.
We've just shown you how to set up, run and analyze a time-lapse imaging experiment to determine the my timing of mitotic progression of cells following sir a transfection. When doing this procedure, it's important to be as gentle to the cells as possible by maintaining the proper temperature and CO2 concentrations and by limiting the amount of light to which the cells are exposed. So that's it.
Thanks for watching and good luck with your experiments.
