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.