Name: assessment of neuronal viability using fluorescein diacetate-propidium iodide double staining in cerebellar granule neuron culture
Uploaded: 2017-05-10T14:00:00
Description: Watch this Scientific Journal Video about Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture at JoVE.com

This work was supported by grants from the Natural Science Foundation of Zhejiang Province (LY15H310007), the Applied Research Project on Nonprofit Technology of Zhejiang Province (2016C37110), the National Natural Science Foundation of China (U1503223, 81673407), the Ningbo International Science and Technology Cooperation Project (2014D10019), the Ningbo Municipal Innovation Team of Life Science and Health (2015C110026), the Guangdong Provincial International Cooperation Project of Science and Technology (No. 2013B051000038), the Shenzhen Basic Research Program (JCYJ20160331141459373), the Guangdong-Hong Kong Technology Cooperation Funding Scheme (GHP/012/16GD), the Research Grants Council of Hong Kong (15101014), Hong Kong Polytechnic University (G-YBGQ), and the K. C. Wong Magna Fund at Ningbo University.

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 &#181;m.
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This protocol was modified from procedures that have been described previously6,7. 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<sup class="x...

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 glutamate1,2. 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. However, glial cells respond differently to neurotoxins as compared to neurons, leading to a bias in the neuronal viability measured by colorimetric assays3.
In viable cells, Fluorescein diacetate (FDA) can be converted into fluorescein by esterase. Propidium Iodide (PI) can interact with the DNA after penetrating dead cells and can be used to indicate apoptosis within the culture. Therefore, FDA-PI double staining can simultaneously evaluate viable cells and dead cells, suggesting that the cell viability can be measured more accurately by combining both colorimetric methods. Moreover, by adding Hoechst, a blue fluorescent stain for nuclei, the accuracy of cell viability could be further improved. The protocol presented here describes FDA-PI double staining and FDA-PI-Hoechst triple staining, which can be used to accurately analyze neuronal viability in primary cultured CGNs.
This protocol takes advantage of visualizing and distinguishing CGNs and glial cells by their different sizes and shapes. After staining, the numbers of viable neurons and dead neurons are counted from representative images taken by fluorescent microscopy. The large-size glial cells are excluded by the comparison of typical CGNs taken under fluorescent mode with those taken under phase contrast mode. A similar strategy can be performed to measure neuronal viability in mixed cell cultures containing neurons and glial cells, such as primary cortical cultures and hippocampal cultures.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma</td>
			<td>P2636</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytosine &#946;-D-Arabinofuranoside</td>
			<td>Sigma</td>
			<td>C1768</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10099141&#160;</td>
			<td>high quality FBS is essential for culture</td>
		</tr>
		<tr>
			<td>100&#215; glutamine</td>
			<td>Gibco</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>100&#215; anti-biotic</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>BME medium</td>
			<td>Gibco</td>
			<td>21010046&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein diacetate</td>
			<td>Sigma</td>
			<td>F7378</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Yesen</td>
			<td>40731ES10</td>
			<td></td>
		</tr>
		<tr>
			<td>rabbit Anti-GAP43 antibody</td>
			<td>Abcam</td>
			<td>ab75810</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse Anti-GFAP antibody</td>
			<td>Cellsignaling</td>
			<td>3670</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sangon Biotech</td>
			<td>A602440</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>Sigma</td>
			<td>T4665</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse&#160;</td>
			<td>Sigma</td>
			<td>D5025</td>
			<td></td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T9003</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Volac</td>
			<td>Z310727</td>
			<td>burn&#160; the tip round before use</td>
		</tr>
		<tr>
			<td>12-well cell culture plates</td>
			<td>TPP</td>
			<td>Z707783</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture plates</td>
			<td>TPP</td>
			<td>Z707759</td>
			<td>high quality cell culture plate&#160; is essential for culture</td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Millipore</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet 5 ml</td>
			<td>Excell Bio</td>
			<td>CS017-0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet 10 ml</td>
			<td>Excell Bio</td>
			<td>CS017-0004</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissect microscope</td>
			<td>Shanghai Caikang</td>
			<td>XTL2400</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo Scientific&#160;</td>
			<td>311</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope&#160;</td>
			<td>Nikon</td>
			<td>TI-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence filter and emmision cubes</td>
			<td>Nikon</td>
			<td>B-2A, G-2A, UV-2A</td>
			<td></td>
		</tr>
		<tr>
			<td>Photo software</td>
			<td>Nikon</td>
			<td>NIS-Elements</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphics editor softeware</td>
			<td>Adobe</td>
			<td>Photoshop CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Image process softeware</td>
			<td>NIH</td>
			<td>ImageJ</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of neuronal viability using fluorescein diacetate-propidium iodide double staining in cerebellar granule neuron culture

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.

The dual immunostaining of GAP43 (red) and GFAP (green) was used to analyze the shape of neurons and glial cells, respectively2,5. 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...

Watch this Scientific Journal Video about Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture at JoVE.com

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

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.

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

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