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 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.
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.
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.
2. Preparation of Dissection Solutions
NOTE: Do this 1 d prior to the dissection.
3. Coating the Cell Culture Plates
NOTE: Do this 1 d prior to the dissection.
4. Processing Tissues for 8 day old Sprague-Dawley Rats.
5. Addition of Ara-C and D-glucose during Culturing
6. FDA-PI Double Staining and FDA-PI-Hoechst Triple Staining in CGN Culture
7. Assessment of Neuronal Viability
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 viability. Therefore, FDA-PI and FDA-PI-Hoechst staining, which can distinguish glial cells from neurons, are advantageous for the accurate evaluation of neuronal viability in CGN culture.
The low-potassium challenge was used to induce neuronal death in a CGN culture. The representative images show the CGN culture challenged with a low-potassium medium (5K), normal medium (25K), or original medium, as analyzed by FDA-PI double staining (Figure 2A) or FDA-PI-Hoechst triple staining (Figure 2B). Neuronal viability measured by various methods is presented in Figure 3 (mean ±SEM). The cell viabilities measured by FDA-PI double staining in the control, 25K, and 5K groups were 99.8 ±4.2%, 98.2 ±2.9%, and 43.9 ±8.6%, respectively (Figure 3A). The cell viabilities measured by FDA-PI-Hoechst triple staining in the control, 25K, and 5K groups were 99.8 ±1.6%, 96.7 ±4.4%, and 48.3 ±4.4%, respectively (Figure 3B). The cell viabilities measured by MTT assay in the control, 25K, and 5K groups were 102.1 ±3.9%, 96.5 ±1.7%, and 57.5 ±5.7%, respectively (Figure 3C). The percentages of lactic dehydrogenase (LDH) release in the control, 25K, and 5K groups were 100.0 ±5.5%, 94.5 ±11.2%, and 202.1 ±15.3%, respectively (Figure 3D). Neuronal viability could not be directly calculated by the LDH assay2. The MTT assay assesses the activity of NADPH-dependent cellular oxidoreductase, which reflects the number of viable cells2. However, this method could not distinguish glial cells from neurons when used to measure neuronal viability in CGN culture. By using FDA-PI and FDA-PI-Hoechst staining, the number of glial cells could be excluded, and neuronal viability could be accurately measured. Moreover, the neuronal viability in 5K medium-treated CGNs measured by FDA-PI or FDA-PI-Hoechst staining was slightly smaller than that measured by MTT assay. This might be because most glial cells that were not sensitive to 5K medium-induced neurotoxicity were excluded by FDA-PI or FDA-PI-Hoechst staining, but not by the MTT assay.
Figure 1: Both Small Neurons and Large, Irregular Glial Cells are Present in CGN Culture. At day 8 in vitro, CGNs were dual immunostained with GAP43 (red) and GFAP (green) for neurons and glial cells, respectively. The phase contrast images show the morphology of the cells. Traditional assays for cell vitality cannot distinguish glial cells from neurons when used to measure neuronal viability. Therefore, FDA-PI and FDA-PI-Hoechst staining, which can distinguish glial cells from neurons, are advantageous for the accurate evaluation of neuronal viability in CGN culture. Scale bar: 100 µm. Blue arrow: typical neurons; white arrow: typical glial cells. Please click here to view a larger version of this figure.
Figure 2: FDA-PI Double Staining and FDA-PI-Hoechst Triple Staining Demonstrate Low-potassium-induced Neuronal Death in CGN Culture. At day 8 in vitro, CGN cultures were switched to 5K or 25K medium. The medium in the control cultures was not changed. After 24 h of challenge, the CGN cultures were assayed by (A) FDA-PI double staining or (B) FDA-PI-Hoechst triple staining. Scale bar = 100 µm. Arrow: typical glial cells. Please click here to view a larger version of this figure.
Figure 3: Quantification of Neuronal Viability in CGN Culture. At day 8 in vitro, CGN cultures were switched to 5K or 25K medium. The medium in the control cultures was not changed. After 24 h of challenge, cell viability was analyzed by (A) FDA-PI double staining, (B) FDA-PI-Hoechst triple staining, and (C) MTT assay. (D) The LDH release was analyzed by LDH assay. The data are expressed as the means ±SEM. **p <0.01 versus control (ANOVA, Tukey's test). Please click here to view a larger version of this figure.
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 maturation9. In this study, Ara-C was used to minimize the growth of glial cells, although there was still about a 1 – 5% presence of glial cells in the CGN culture. This problem is also presented in other groups' studies10. In CGN culture, the sizes and shapes of glial cells are largely different from neurons. The diameter of CGNs is about 10 µm, and mature CGNs have rich processes that connect the neurons. However, the diameter of glial cells in culture is relative large, about 30-100 µm, so it is easy to distinguish glial cells from neurons in images. Therefore, one advantage of FDA-PI and FDA-PI-Hoechst staining is to accurately measure neuronal viability in primary neuronal cultures in which non-neuronal cell growth is not prevented by the culture techniques.
Low-potassium-induced apoptosis in CGN cultures, with 25K medium-treated cells as the control, represents an excellent model to study neuronal apoptosis. Many groups, including our lab, have used this classic model study the underlying apoptotic mechanisms of neurons2,11. Therefore, this model was used to induce neuronal death. The concentration of dyes and the duration of staining used in this protocol have been optimized. Low concentrations of dyes and short durations of staining can cause insufficient staining, leading to the inaccurate counting of dead neurons. However, high concentrations of dyes and prolonged durations of staining can further induce neuronal death and cause a bias in the estimation of cell viability. Therefore, the images should be taken as soon as possible after the dyes are added. In the FDA-PI double staining images, small FDA-positive cells are marked as viable neurons. However, cell bodies tend to group up in CGN culture. Therefore, it can be hard to separate viable cells by FDA staining. In this study, Hoechst, a nuclear stain, was used to improve the accuracy. Small cells with FDA-positive cell bodies and Hoechst-positive nuclei can be regarded as viable neurons in FDA-PI-Hoechst triple staining images.
Compared to colorimetric cytotoxicity assays, FDA-PI and FDA-PI-Hoechst staining require more time for the evaluation of neuronal viability in CGN culture. Therefore, the current protocol might not be suitable to screen neuroprotective drugs. However, this limitation can be resolved by using a high-content imaging system. Another limitation of this technique is that CGNs and glial cannot be discriminated by PI. However, because neurons are more susceptible to neurotoxins than glial cells, PI-positive dead cells are mainly neurons12.
Besides FDA, PI, and Hoechst, other dyes, such as MTT, SYTO13, and SYBR14, are also used to measure cell viability13,14. For the MTT assay, glial cells that are not sensitive to neurotoxins can convert MTT to formazan, leading to the over-estimation of neuronal viability. SYTO13 is cell-permeable and has a high green fluorescent yield when bound to DNA or RNA. A previous study suggested that SYTO13 staining mainly indicates apoptotic cells in CGN culture but does not distinguishes glial cells from neurons13. SYBR14 is a membrane-permanent nucleic acid dye. SYBR14-PI double staining is used to measure cell viability because both dyes label DNA, thereby circumventing the ambiguity14. However, SYBR14-PI double staining cannot effectively distinguish glial cells from neurons in CGN culture. Therefore, FDA-PI and FDA-PI-Hoechst staining, which can distinguish glial cells from neurons, are advantageous for the accurate evaluation of neuronal viability in CGN culture.
Finally, FDA-PI and FDA-PI-Hoechst staining are not limited to the analysis of neuronal viability in CGN cultures. Many mixed cell cultures (e.g., primary cortical cultures and primary hippocampal cultures) also contain neurons and glial cells. Therefore, a similar strategy can be applied to those mixed cell cultures to accurately analyze neuronal viability.
The authors have nothing to disclose.
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.
Poly-L-lysine | Sigma | P2636 | |
D-glucose | Sigma | G8270 | |
Cytosine β-D-Arabinofuranoside | Sigma | C1768 | |
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 | |
Hoechst 33342 | Yesen | 40731ES10 | |
rabbit Anti-GAP43 antibody | Abcam | ab75810 | |
mouse Anti-GFAP antibody | Cellsignaling | 3670 | |
Bovine serum albumin | 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 process softeware | NIH | ImageJ |