Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Summary

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

Abstract

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

Introduction

Colorimetric cytotoxicity assays, such as the 3- (4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay, are commonly used to measure cell viability in vitro. Primary cultured Cerebellar Granule Neurons (CGNs) from rats are sensitive to various neurotoxins, including 1-methyl-4-phenylpyridinium ion, hydrogen peroxide, and glutamate^1,2. Therefore, CGN cultures can be used as an in vitro model in the field of neuroscience. CGN cultures may contain a variety of cells, including neurons and glial cells, which can account for about 1% of the total cells in the CGN culture. H....

Protocol

All procedures followed the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1996) and were approved by the Institutional Animal Care and Use Committee (IACUC) at Ningbo University (SYXK-2008-0110).

1. Preparation of Solutions and Culture Media

Note: Reagents and stocks need to be prepared under sterile conditions. Sterilize by filtering using a filter with a pore size of 0.22 µm.

1. Prepare Poly-L-Lysine (PLL) stock solution by adding 5 mg of PLL  to 10 mL of double-distilled water (ddH₂O). Sterilize by filteri....

Results

The dual immunostaining of GAP43 (red) and GFAP (green) was used to analyze the shape of neurons and glial cells, respectively^2,5. Both neurons and glial cells are present in CGN culture. The GFAP-positive glial cells are large and irregular in shape, as indicated by the arrows in the images (Figure 1). Traditional assays for cell vitality cannot distinguish glial cells from neurons when used to measure neuronal v.......

Discussion

This protocol was modified from procedures that have been described previously^6,7. Researchers have spent time trying to reduce non-neuronal cell growth by improving the conditions when culturing primary neurons in vitro⁸. However, even with improved culture conditions, some glial cells remain. Moreover, non-neuronal cells are necessary in primary neuronal cultures because they help with neuronal growth and maturation

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Poly-L-Lysine (PLL)	Sigma	P2636
D-glucose	Sigma	G8270
Cytosine β-D-Arabinofuranoside	Sigma	C1768
Fetal Bovine Serum (FBS)	Gibco	10099141	high quality FBS is essential for culture
100x glutamine	Gibco	25030081
100x antibiotics	Gibco	15240062
Basal Medium Eagle (BME)	Gibco	21010046
Fluorescein diacetate (FDA)	Sigma	F7378
Propidium Iodide (PI)	Sigma	P4170
Hoechst 33342	Yesen	40731ES10
rabbit Anti-GAP43 antibody	Abcam	ab75810
mouse Anti-GFAP antibody	Cellsignaling	3670
Bovine Serum Albumin (BSA)	Sangon Biotech	A602440
Trypsin	Sigma	T4665
DNAse	Sigma	D5025
Soybean trypsin inhibitor	Sigma	T9003
Pasteur pipette	Volac	Z310727	burn the tip round before use
12-well cell culture plates	TPP	Z707783
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
Dissect microscope	Shanghai Caikang	XTL2400
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 processing software	NIH	ImageJ