The overall goal of this procedure is to demonstrate live imaging and simple quantification methods to analyze embryonic epithelial cell morphology. This is accomplished by first getting all the tools and apparatus ready. The second step of the procedure is to excise X explan from frog embryos and house them in chambers for long-term culture.
The third step of the procedure is to collect high resolution images of epithelial cells in real time. The final step of the procedure is to quantify morphological details of epithelial cells using Image J.Ultimately, results can be obtained that show tracked cell areas and membrane intensities through high resolution confocal microscopy. Hi, I'm Sagar Joshi from Davidson Laboratory in the Department of Bioengineering at University of Pittsburgh.
Today we will show you a procedure for live cell imaging and quantitative analysis of Xenos lavia embryonic epithelial cells. We use this procedure to study realtime cell cytoskeletal changes. So let's get started.
To prepare chambers for culturing animal cap caps, use silicone grease to seal a custom acrylic chamber or small nylon washer to a large 45 by 50 millimeter cover glass. Add one milliliter of 1%BSA in one third XMBS to the chamber. Coat small cover slip fragments with the same solution in incubate for two to four hours at room temperature or overnight at four degrees Celsius.
To reduce adhesion of the X explan to the glass, rinse and fill the chamber with DFA medium until just before transferring in The xs, rinse the cover slip fragments in the same manner. Then immerse them in a dish of DFA culture embryos that were micro injected at the one to four cell stage with the desired MR NA to the desired stage in one third XMBS. Use a transfer pipette with a slanted opening to transfer the embryos to a dish of DFA under a dissecting stereo microscope.
Use forceps to ize the embryos to excise an animal CapEx plant. Use a hair loop to support an embryo in one position. Use a hair knife to make a small incision in the animal pole.
Make repeated smooth flick cuts to extend the incision and remove the cap ectoderm. Excise three or four x explan in the same manner. And use a transfer pipette to move them to the prepared culture chamber.
Using the hair loop position each X explan so that the epithelium faces the bottom of the chamber. Using forceps. Hold a cover slip fragment at its center.
Dip the ends in silicone grease. Place one fragment over each X explan. Lightly fix each fragment in place with small dabs of silicone grease.
Taking care not to smash the X explan with excessive force completely. Fill the chamber with DFA coat the top edges of the chamber with silicone grease. Then seal the top with a 24 x 40 millimeter cover slip.
Adjust the level of the DFA within the chamber to reduce air bubbles and use tissue to blot away any overflow. The X plants can now be cultured long-term in the sealed chamber for a day at room temperature, move the 20 x objective of a confocal microscope into place. Position the chamber containing the X explan onto the microscope stage and place small balancing weights on the chamber to hold it in position under bright field.
Adjust the focus and use course positioning to visualize the apical ends of the superficial cells. Switch to fluorescence and a higher power objective. To collect single images or time-lapse movies of the explan epithelium, tune the acquisition to attain the best possible image.
Refer to the written portion of this protocol for tips on tuning and imaging. Keeping the laser power as low as possible. Capture images or time-lapse movies of the x explan epithelium.
Refer to the written portion of the protocol for suggestions on data collection. Confocal image files can be mined for data in various ways. Here are two examples of how to analyze data files using Image J analysis software with bio formats plugin.
To measure the cell area of an epithelial cell, pull down the analyze menu and click set measurements. Select the selection tool from the toolbar and outline the cell in the analyze menu, select tools, and then ROI manager. To open the regions of interest manager press control t.
To add the outline to the ROI manager, select and add as many outlines as desired from the image. Then click save to save the ROI set in the ROI manager. Click measure.
The results window will now show the measured areas of each ROI. To measure the intensity of a cell membrane, select the straight line tool from the image J toolbar. Draw a perpendicular line across the membrane.
Press CTRL T as in the previous example. To add the selection to the ROI manager, pull down the analyze menu and click on plot profile to generate a plot of relative intensities across the line. To save the graph as an image, click save in the new plot window that appears.
Pull down the list menu to file, then save as We have just shown you how to live image and quantify embryonic epithelial cells from excised animal CapEx explan. While doing this procedure, it is important to remember to add antibiotic and antibiotic to the DFA. To discourage bacterial and fungal growth, take extra care while pressing explants under the cover slips because more than required pressure can smash the ex explan.
It is also important to remember to have optimal settings on the confocal microscope to get maximal quality image data. So that's it. Thank you for watching and good luck with your experiments.
