The overall goal of this procedure is to accurately measure cell viability in a cerebellar granule neuron culture. This method can help to answer key questions in the field of neuroscience and neuropharmacology. The main advantage of this technique is that we can distinguish unwanted glial cells from neurons in MISTA cell cultures.
Demonstrating the procedures will be Lin Jiajia, Wang Jialing, Xu Dilin, three undergraduate students from my lab. Begin dissecting the heads of eight-day-old rat pups by first inserting scissors into the foramen magnum and cutting the two sides of the skull from the ears to the eyes. Then, use forceps to lift the skull.
Use forceps to isolate the cerebellum, and place it into a 35-millimeter dish containing dissection solution. Transfer the plate to the stage of the dissecting microscope and use two pairs of forceps to remove the meninges and blood vessels. Use a blade to chop the tissues, and then transfer the tissue into a centrifuge tube containing 30 milliliters of dissection solution.
Centrifuge for five minutes at room temperature at 1, 500 x g. Following the centrifugalization, aspirate the supernatant and save the pellet. Next, add Trypsin solution to the pellet and resuspend by shaking gently.
Then place the tube in a 37 degree celsius water bath for 15 minutes. After 15 minutes, add a solution of DNase One, soybean Trypsin inhibitor, and magnesium sulfate, and shake. Then centrifuge for five minutes at room temperature at 1, 500 x g.
After aspirating the supernatant, resuspend the pellet in a less dilute DNase One, soybean Trypsin inhibitor, and magnesium sulfate solution. Next, prepare two 15-milliliter tubes. Place half of the cells in each tube, and pipette both up and down 60 to 70 times using a cotton-plugged Pasteur pipette to homogenize the cells.
After homogenization, add three milliliters of magnesium sulfate and calcium chloride solution to each 15-milliliter tube. Allow the tubes to sit at room temperature for 10 minutes. Once the time has elapsed, carefully remove the supernatant with a cotton-plugged Pasteur pipette and transfer to new 15-milliliter tubes.
Centrifuge for five minutes at room temperature and 1, 500 x g. Add culture medium to the pellet to dilute to a cell density of around 1.5 x 10 to the sixth cells per milliliter. Pipette the cells into a 6-well plate, and culture in the incubator at 37 degrees celsius and 5%carbon dioxide.
After 24 hours, add AraC stock solution to a final concentration of one millimolar to inhibit the growth of the glial cells before returning the plates to the incubator. Then, on day seven, add 50 microliters to D-glucose stock solution to a final concentration of one millimolar. Begin the staining by mixing fluorescein diacetate to a final concentration of 10 micrograms per milliliter, and propidium iodide to a final concentration of 50 micrograms per milliliter and 10 milliliters of PBS.
Mix the FDA/PI solution by vortexing and place on ice. Place the culture plate on ice. Carefully aspirate the culture medium without touching the cells with the pipette tip, then slowly add cold PBS.
Aspirate the PBS, then add cold FDA/PI, or FDA/PI Hoechst working solution. Put the plate on ice for five minutes. Next, after aspirating the FDA/PI, or FDA/PI/Hoechst working solution, add cold PBS to each well.
Make sure that the fluorescent microscope is set up correctly. Detect the fluorescent emissions for FDA, PI, and Hoechst at 520, 620, and 460 nanometers, respectively, using an exposure time between 100 and 300 milliseconds and an analog gain of 2.8 x. After collecting fluorescence images, take an image under normal light using the phase contrast mode.
For FDA/PI double staining, overlay the images of the cells by dragging the FDA positive layer on top of the PI positive layer in graphics editing software. Adjust the opacity of the FDA positive layer by entering 50%in the Opacity field of the Layers panel. Merge two layers by opening the Layer menu and clicking on the Merge Visible button.
Set the contrast of the overlay images by entering 50 in the Contrast field under Image, Adjustments, Brightness/Contrast. Check that there are no FDA and PI double positive cells in the overlay images. For FDA/PI/Hoechst triple staining, overlay the images of the cells by again dragging the FDA positive layer on the PI positive layer, adjusting the opacity of the FDA positive layer and merging the images as before.
Then drag the Hoechst positive layer on the merged layer. Adjust the opacity of the Hoechst positive layer by entering 50%in the Opacity field of the Layers panel, and merge the two layers by opening the Layer menu and clicking on the Merge Visible button. Visually distinguish large, irregular glial cells from cerebellar granular neurons by comparing the images taken under fluorescent mode with those taken under phase contrast mode.
The following images show that both small neurons and large, irregular glial cells are present in CGN culture. This image shows GAP-43 amino staining of a CGN culture at eight days in vitro. The blue arrows indicate neurons.
Here, GFAP amino staining of glial cells is shown. The white arrow indicates a glial cell. This phase contrast image shows the morphology of the cells.
FDA/PI hook staining which can distinguish glial cells from neurons is advantageous for the accurate evaluation of neuronal viability in CGN culture. This is a merged image of triple-stained cells grown in normal growth medium. The arrow shows a typical glial cell.
Here, a similar culture has been treated with medium containing low potassium, and the arrow again indicates a typical glial cell. Also, note the appearance of red propidium iodide staining. Once mastered, this technique can be done in two hours if it is performed properly.
A similar strategy can be applied to other mixed cell cultures, such as primary cortical cultures, and primary hippocampal cultures to accurately analyze neuronal viability.
