Assessing Neuronal Viability in a Cerebellar Neuron Culture via Double Staining

Abstract

Source: Jiajia, L., et al. Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture. J. Vis. Exp. (2017).

This video demonstrates the method of identifying neurons from a mixed culture of cerebellar granule neurons and glial cells using dual staining with fluorescein diacetate (FDA) and propidium iodide (PI), where live neurons appear green and dead neurons appear red.

Protocol

1. FDA-PI Double Staining and in Cerebellar Granule Neuron (CGN) Culture

1. Prepare fluorescein diacetate-propidium iodide (FDA-PI) working solution by adding 20 µL of FDA stock solution (final concentration: 10 µg/mL) and 50 µL of PI stock solution (final concentration: 50 µg/mL) in 10 mL of phosphate-buffered saline (PBS). Mix by vortexing and place this on ice.
   NOTE: Freshly prepare the FDA-PI working solution working solution just before use.
2. Take the cell culture plate out of the incubator. Put it on ice.
3. Aspirate the culture medium and replace it with cold PBS.
   NOTE: The change of solution must be done slowly and carefully. Avoid touching the cells with pipette tips.
4. Aspirate the cold PBS and replace it with cold FDA-PI (500 µL/well for 12-well cell culture plates or 1 mL/well for 6-well cell culture plates). Leave on ice for 5 min.
5. Aspirate the FDA-PI and add cold PBS (100 µL/well for 12-well cell culture plates or 50 µL/well for 6-well cell culture plates).
   NOTE: Cells should not be allowed to dry when taking images.
6. Take images using fluorescent microscopy. Use an excitation filter with a pass band of 450 - 490 nm. Detect the fluorescence emissions for FDA and PI at 520 and 620 nm, respectively. Under the same conditions, take an image under normal light using the phase contrast mode of fluorescent microscopy.
   NOTE: Take images within 15 minutes after FDA-PI. The exposure time is 100-300 ms, and the analog gain is 2.8x.

2. Assessment of Neuronal Viability

1. For FDA-PI double staining, overlay the images of the cells detected after filtering by different fluorescence filters by dragging the FDA-positive layer on the PI-positive layer in graphics editor software.
   1. Adjust the opacity of the FDA-positive layer by entering "50%" in the "Opacity" field of the "Layers" panel. Merge two layers by clicking on the "Merge Visible" button in the "Layer" menu. Set the contrast of the overlay images by entering "50" in the "Contrast" field under "Image"| "Adjustments" | "Brightness/Contrast." Make sure that there are no FDA and PI double-positive cells in the overlay images.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fetal bovine serum	Gibco	10099141	high quality FBS is essential for culture
100× glutamine	Gibco	25030081
100× anti-biotic	Gibco	15240062
BME medium	Gibco	21010046
Fluorescein diacetate	Sigma	F7378
Propidium iodide	Sigma	P4170
Rabbit Anti-GAP43 antibody	Abcam	ab75810
Mouse Anti-GFAP antibody	Cell Signaling	3670
Pasteur pipette	Volac	Z310727	burn the tip round before use
6-well cell culture plates	TPP	Z707759	high quality cell culture plate is essential for culture
Filter	Millipore	SLGP033RB
Pipet 5 ml	Excell Bio	CS017-0003
Pipet 10 ml	Excell Bio	CS017-0004
CO2 Incubator	Thermo Scientific	311
Fluorescence microscope	Nikon	TI-S
Fluorescence filter and emmision cubes	Nikon	B-2A, G-2A, UV-2A
Photo software	Nikon	NIS-Elements
Graphics editor softeware	Adobe	Photoshop CS
Image process software	NIH	ImageJ