Assessing Neuronal Viability in a Cerebellar Neuron Culture via Double Staining

Begin with a culture plate treated with an anti-mitotic agent that inhibits glial cell growth, leaving primarily cerebellar granule neurons and a small number of glial cells.

Remove the medium and slowly add cold phosphate-buffered saline or PBS to maintain cell viability.

Replace the buffer with a cold staining mixture and incubate.

This mixture contains fluorescein diacetate or FDA, a non-fluorescent compound, and propidium iodide or PI, a membrane-impermeable fluorescent dye.

FDA diffuses into cells, where the functional esterase in live cells converts it into a green fluorescent compound.

PI enters into dead cells through damaged membranes, binds to DNA, and emits red fluorescence.

Remove the staining solution and add cold PBS to prevent the cells from drying.

Obtain fluorescent images for FDA and PI stains and a phase-contrast image. Merge the FDA and PI images.

Then compare this fluorescence image with the phase-contrast image, which helps to exclude the large glial cells.

Red fluorescence indicates neuronal death, whereas green fluorescence signifies neuronal viability.

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 in 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 fluorescence 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 2.8X. 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 overlaid 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.