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 ...

The overall goal of this procedure is to accurately measure cell viability in a cerebellar granule neuron culture. This method can help to answer key questions in the field of neuroscience and neuropharmacology. The main advantage of this technique is that we can distinguish unwanted glial cells from neurons in MISTA cell cultures.Demonstrating the procedures will be Lin Jiajia, Wang Jialing, Xu Dilin, three undergraduate students from my lab. Begin dissecting the heads of eight-day-old rat pups by first inserting scissors into the foramen magnum and cutting the two sides of the skull from the ears to the eyes. Then, use forceps to lift the skull.Use forceps to isolate the cerebellum, and place it into a 35-millimeter dish containing dissection solution. Transfer the plate to the stage of the dissecting microscope and use two pairs of forceps to remove the meninges and blood vessels. Use a blade to chop the tissues, and then transfer the tissue into a centrifuge tube containing 30 milliliters of dissection solution.Centrifuge for five minutes at room temperature at 1, 500 x g. Following the centrifugalization, aspirate the supernatant and save the pellet. Next, add Trypsin solution to the pellet and resuspend by shaking gently.Then place the tube in a 37 degree celsius water bath for 15 minutes. After 15 minutes, add a solution of DNase One, soybean Trypsin inhibitor, and magnesium sulfate, and shake. Then centrifuge for five minutes at room temperature at 1, 500 x g.After aspirating the supernatant, resuspend the pellet in a less dilute DNase One, soybean Trypsin inhibitor, and magnesium sulfate solution. Next, prepare two 15-milliliter tubes. Place half of the cells in each tube, and pipette both up and down 60 to 70 times using a cotton-plugged Pasteur pipette to homogenize the cells.After homogenization, add three milliliters of magnesium sulfate and calcium chloride solution to each 15-milliliter tube. Allow the tubes to sit at room temperature for 10 minutes. Once the time has elapsed, carefully remove the supernatant with a cotton-plugged Pasteur pipette and transfer to new 15-milliliter tubes.Centrifuge for five minutes at room temperature and 1, 500 x g. Add culture medium to the pellet to dilute to a cell density of around 1.5 x 10 to the sixth cells per milliliter. Pipette the cells into a 6-well plate, and culture in the incubator at 37 degrees celsius and 5%carbon dioxide.After 24 hours, add AraC stock solution to a final concentration of one millimolar to inhibit the growth of the glial cells before returning the plates to the incubator. Then, on day seven, add 50 microliters to D-glucose stock solution to a final concentration of one millimolar. 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 and 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 fluorescent 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 2.8 x. 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 overlay 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.The following images show that both small neurons and large, irregular glial cells are present in CGN culture. This image shows GAP-43 amino staining of a CGN culture at eight days in vitro. The blue arrows indicate neurons.Here, GFAP amino staining of glial cells is shown. The white arrow indicates a glial cell. This phase contrast image shows the morphology of the cells.FDA/PI hook staining which can distinguish glial cells from neurons is advantageous for the accurate evaluation of neuronal viability in CGN culture. This is a merged image of triple-stained cells grown in normal growth medium. The arrow shows a typical glial cell.Here, a similar culture has been treated with medium containing low potassium, and the arrow again indicates a typical glial cell. Also, note the appearance of red propidium iodide staining. Once mastered, this technique can be done in two hours if it is performed properly.A similar strategy can be applied to other mixed cell cultures, such as primary cortical cultures, and primary hippocampal cultures to accurately analyze neuronal viability.

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