The overall goal of this imaging analysis is to observe vesicle motility in neurons and quantify it easily. This approach enables one to analyze membrane trafficking in neurons in a straightforward and time-effective manner. To begin this procedure, transfer the uterus to a new Petri dish with 70%ethanol.
Next, transfer the uterus to another Petri dish with HBSS. Then remove the fetuses from the uterus using sterilized dissecting forceps and surgical scissors. After that place them in a new Petri dish with HBSS.
Under a stereo microscope remove the skin and skulls and take out the brains using the sterilized dissecting forceps. Then, transfer them to another Petri dish with HBSS. Remove the meninges using sterilized dissecting forceps.
Then trim off the extra regions. Next, transfer the cortices to a new Petri dish with HBSS. Collect all the cortices using a disposable dropper or a 25 milliliter pipette and place them into a 15 milliliter tube.
Following that, remove the HBSS by pipetting. Then, add three milliliters of cerebral cortical enzyme solution to the tube containing the cortices. Place the tube in a water bath and incubate it at 37 degrees Celsius for five minutes.
After that add another three milliliters of cerebral cortical enzyme solution into the tube containing the cortices and incubate them at 37 degrees Celsius in a water bath for an additional five minutes. After five minutes remove the cerebral cortical enzyme solution and add five milliliters of DMEM containing 10%fetal bovine serum. Dissociate the cortices by gently pipetting up and down using a five milliliter pipette with a sterile P1000 tip.
Then pipette the cortices again using a five milliliter pipette with a P200 tip. Put a 40 micrometer nylon cell strainer on a 50 milliliter tube and filter the suspension to remove the debris. Subsequently, centrifuge the suspension at 420 times g for five minutes at room temperature, and then remove the DMEM medium.
Afterward, add five milliliters of cerebral cortical culture medium and gently resuspend the cell pellet. Then count the cells using a hemocytometer and adjust the cell concentration using the cerebral cortical culture medium. Next, plate three times 10 to the six cortical neurons in two milliliters of cell suspension for each coded 35 milliliter glass bottom dish.
In this procedure, mix four micrograms of plasmid with 200 microliters of serum-free medium. In a separate tube dilute eight microliters of the transfection reagent in 200 microliters of serum-free medium. Then, incubate the solution for five minutes at room temperature.
After five minutes mix the plasmid solution and the transfection reagent solution and incubate the mixture for 20 minutes at room temperature. Following that, add the mixture to the coded glass bottom dishes. Incubate the neurons at 37 degrees Celsius for 30 minutes.
Afterward, replace the medium with two milliliters of cerebral cortical culture medium and incubate the neurons at 37 degrees Celsius for one to two days. For imaging, use a fluorescence microscope equipped with a CCD camera with a 40X objective lenses. Keep the temperature at 37 degrees Celsius using an incubation system.
Acquire neuron images at one frame every five seconds over a 100-second period controlled by the image acquisition software. To analyze the images, open all the sequential images in ImageJ software with Manual Tracking. To combine the sequential images choose Image, then Stacks, followed by Images to Stack and then click OK.After that, choose Plugins then Manual Tracking.
A Tracking window will pop up. Click on the checkbox of Show Parameters and define the tracking parameters in the Parameters section. Then click Add track to start a new track.
After determining the vesicles of interest click on the center of the signals in the sequential images to record the xy coordinates. Repeat this procedure to collect the xy coordinates of the vesicles from all sequential images, each image automatically advances to the successive image after the reference point is selected. This image shows the motility of LAMP-EGFP containing vesicles in the dendrites of the mouse cortical neurons after two days of transfection, started at four DIV.
The inset shows the time-lapse image of the LAMP-EGFP positive vesicles. The red arrowhead indicates a moving vesicle and the blue arrowhead indicates a resting vesicle. The orange arrowheads indicate higher fluorescent signals.
Here is the time-dependent X-axis position of the LAMP-EGFP positive vesicles. The vesicle in the red circle, labeled b, is moving and the vesicle in the blue circle, labeled a, is resting. This bar graph indicates the total distance of LAMP-EGFP movement.
And this bar graph indicates the average motility distance of a LAMP-EGFP containing vesicles.
