2次元培養系を用いた主な大脳皮質細胞のイメージングをライブします。

The overall goal of this experiment is to observe cell behaviors during lineage progression from neural stem cells to neurons or glial cells. This method can help answering questions in the neurodevelopmental field such as the role of symmetric and asymmetric cell divisions, cell cycle lengthening and cell growth rates. The main advantage of this technique is that it allows the observation of cell lineage for long periods.

This permits the observation of switch from neurogenesis to glial genesis within a single lineage and a direct comporation of sublinear neural and glial progenitors. Begin by removing the brains from 5 to 10 e14 embryos in a Petri dish with cold dissection medium. Use forceps to carefully pull out the skin and skull and isolate the brains.

While working under the stereo microscope, use dissection forceps to remove the meninges. Then, split the telencephalon in the middle to separate the hemispheres. To isolate the dorsolateral telencephalon, cut along the dorsomedial curve and pallio and subpallium boundary.

Transfer the dorsolateral telencephalons to a 2mL tube with dissection medium on ice until all brains are dissected. After micro-dissection of the dorsolateral telencephalon, place the tissue in a 15mL conical tube. Then centrifuge the tube containing the collected tissue in cold dissection medium for five minutes at four degrees Celsius and 340 times G to precipitate the tissue.

After the spin, remove the supernatant with a pipette and add 1mL of 37 degrees Celsius 0.05%Trypsin-EDTA for chemical digestion. Incubate for 15 minutes at 37 degrees Celsius. Following the incubation, add 2mL of proliferation medium to inhibit Trypsim activity.

Next, polish the tip of a glass Pasteur pipette over a gas burner or bunsen burner for a few seconds to slightly narrow the aperture. Asperate FCS to coat the tip of the pipette. Then mechanically dissociate the cells with the fire polished and FCS coated Pasteur pipette by pipetting up and down while being careful to avoid generating bubbles.

Centrifuge the dissociated cells for five minutes at four degrees Celsius at 340 times G.Remove the supernatant with a pipette, then add one millimeter of proliferation medium and resuspend the cells using a pipette. Remove the PBS from the pretreated plate with Poly-D-Lysine. Then draw a sign on the bottom of the plate that can be used as a reference to determine the zero point.

After resuspending the cells in proliferation medium for the second time, prepare a one to one dilution of the cell suspension using a 0.4%trypan blue solution. Load into counting chamber slides and count the number of unstained viable cells. Non-viable cells will be blue.

Dilute the cells in proliferation medium to get 10 to the sixth cells per millimeter. Add 500 microliters of the cell suspension to each well of a 24 well tissue culture plate and incubate cells at 37 degrees Celsius and 5%carbon dioxide. To image retrovirus transduced cells, place the tissue culture plate into an imaging chamber connected to temperature and carbon dioxide controllers to maintain the cells at constant conditions of 37 degrees Celsius and 5%carbon dioxide.

Select a position in the xy axis to serve as a zero point and calibrate the zero point. Select 10 to 15 positions per well to be imaged at 10 times magnification. Set the software to require phase contrast images every five minutes and fluorescence images every three hours to decrease phototoxicity.

Check and adjust the focus in the first three hours of the experiment as it may change while the temperature equilibrates. After seven days, stop the acquisition and proceed to post-imaging immunocytochemistry. Start the cell tracking software and choose a username.

Click on choose your username and continue. Browse and select the tTt work folder to save lineage trees, statistics and exported images. Next, select the experiment to be analyzed and loaded.

If the images were converted previously by tTt converter, click on log file converter and specify the number of seconds between two consecutive time points in the log file converter window. Click on convert log files for whole experiment. Select and load the desired images manually or using the option displayed in the program.

Select file, open, new colony. Click on tracking, followed by start tracking to start the tracking in a chosen position. A movie window will appear.

Select the cell using the tracking circle and press the zero key. The software will go to the next frame. Keep positioning the tracking circle around the track cell using the mouse and click zero in each frame to add a track mark on the cell.

Use keys one and three to move to either the previous or next frame. To delete a track mark, just click on the correct position to mark and press zero. If the cell has divided, select tracking, stop reason, division and continue tracking one daughter cell by selecting one of the cells in the cell editor window, then select tracking, start tracking.

To track the second daughter cell, select it in the cell editor and press F2.If the cell died during the video microscopy, select apoptosis. If the cell leaves the field of observation or intermingles with neighboring cells precluding tracking, select lost. To stop tracking and continue later, click on interrupt.

In the cell editor window, a tree will be generated during the tracking. To save the lineage tree, select file, save, current tree in the cell editor window. To export the movie with the tracking data, turn off the tracking mode, and in the movie window select export movie.

Set up the format range of frames, number of frames per second and bit rate. Click on start export. The following photomicrographs show phase contrast in GFP fluorescent images of a primary cerebra cortex cell culture.

GFP expression is absent during the first hours of imaging. The number of GFP expressing cells increases over time and the intensity of GFP fluorescence increases as indicated by the yellow arrow in these images. This higher magnification image in the boxed region of the prior image show the GFP expressing cells in detail.

These phase contrast images show an example of cell division. Progenitors cell round up is seen here followed by cell membrane constriction and lastly, mitosis completion. This is an example of a single cell lineage tree.

Colored arrows indicate different modes of cell division, symmetric proliferative division is shown by the yellow arrow. Asymmetric division is indicated by the blue arrow. Symmetric terminal division is shown by the red arrow and X indicates cell death.

Numbers indicate the cell cycle length of the progenitors cells. This technique can be done in one to two hours if it's performed properly. After watching this video, you should have a good understanding of how to show cell behaviors during cell lineage progression for stem cells to neurons or glial cells.