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. 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.

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 filtering. Aliquot and store the PLL stock solution at -20 °C for several months.
2. Prepare Krebs buffer by adding 7.25 g of NaCl, 0.4 g of KCl, 0.14 g of NaH₂PO₄, 2.6 g of D-glucose, and 5.94 g of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid to 100 mL of ddH₂O. Sterilize by filtering. Store the Krebs buffer solution at 4 °C for up to one month.
3. Prepare 3.82% Mg₂SO₄ solution by adding 3.82 g of Mg₂SO₄ to 100 mL of ddH₂O. Sterilize by filtering. Store the Mg₂SO₄ solution at 4 °C for several months.
4. Prepare 1.2% CaCl₂ solution by adding 1.2 g of CaCl₂ to 100 mL of ddH₂O. Sterilize by filtering. Store the CaCl₂ solution at 4 °C for several months.
5. Prepare 2M KCl solution by adding 15 g of KCl to 100 mL of ddH₂O. Sterilize by filtering. Store the KCl solution at 4 °C for several months.
6. Prepare the D-glucose stock solution by adding 1.8 g of D-glucose to 100 mL of ddH₂O. Sterilize by filtering. Aliquot and store the D-glucose solution at 4 °C for up to one month.
7. Prepare Cytosine β-D-Arabinofuranoside (Ara-C) stock solution by adding 0.24 g of Ara-C to 10 mL of ddH₂O. Sterilize by filtering. Aliquot and store the Ara-C stock solution at 4 °C for several months.
8. Prepare 250 mL of culture medium (for 25-30 pups) using 25 mL of Fetal Bovine Serum (FBS), 2.5 mL of 100x glutamine, 2.78 mL of 2M KCl solution, and 2.5 mL of 100x antibiotics in Basal Medium Eagle (BME). Adjust the volume to 250 mL and sterilize by filtering.
9. Prepare 25K medium using 2.5 mL of 100x glutamine, 2.78 mL of 2M KCl solution, and 2.5 mL of 100x antibiotics in BME. Adjust the volume to 250 mL and sterilize by filtering.
10. Prepare 5K medium using 2.5 mL of 100x glutamine and 2.5 mL of 100X antibiotics in BME. Adjust the volume to 250 mL and sterilize by filtering.
    NOTE: The culture medium, 25K medium, and 5K medium need to be freshly prepared
11. Prepare FDA stock solution by adding 5 mg of FDA to 1 mL of acetone. Store the FDA stock solution at 4 °C for long-term storage.
12. Prepare PI stock solution by adding 1 mg of PI to 1 mL of ddH₂O. Store the PI stock solution at 4 °C for several months.
13. Prepare Hoechst stock solution by adding 5 mg of Hoechst 33342 to 1 mL of ddH₂O. Store the Hoechst stock solution at 4 °C for several months.

2. Preparation of Dissection Solutions

NOTE: Do this 1 d prior to the dissection.

1. Prepare the dissection solution by adding 15 mL of Krebs buffer, 1.2 mL of 3.82% Mg₂SO₄ solution, and 0.45 g of Bovine Serum Albumin (BSA) to 150 mL of ddH₂O. Adjust the pH to 7.4 using NaOH.
2. Label 5 x 50 mL tubes as follows: 1, 2, 3, 4, and 5.
3. Place 40 mL of dissection solution into a 50 mL syringe with a filter. Filter 30 mL of dissection solution into tube 1, and 2 mL each to several 35 mm cell culture dishes, which will be used to process tissues.
4. To tube 2, add 12.5 mg of trypsin in 50 mL of dissection solution. Mix completely by vortexing. Sterilize by filtering.
5. To tube 3, add 1.2 mg of DNAse I, 7.8 mg of soybean trypsin inhibitor, and 150 µL of 3.82% Mg₂SO₄ solution in 15 mL of dissection solution. Mix completely by vortexing. Sterilize by filtering.
6. To tube 4, add 5 mL of the solution from tube 3. Add 10.5 mL of dissection solution. Sterilize by filtering.
7. To tube 5, add 100 µL of 3.82% Mg₂SO₄ solution and 15 µL of 1.2% CaCl₂ solution in 12.5 mL of dissection solution. Mix completely by vortexing. Sterilize by filtering.
8. Store all tubes at 4 °C before use.

3. Coating the Cell Culture Plates

NOTE: Do this 1 d prior to the dissection.

1. Add 1 mL of PLL stock solution to 100 mL of ddH₂O (final concentration: 5 µg/mL). Coat plastic 6-well or 12-well cell culture plates (2 mL per well for 6-well cell culture plates or 1 mL per well for 12-well cell culture plates) by adding 5 µg/mL of poly-L-lysine solution.
2. Incubate at room temperature for 1 day (or at 37 °C for 2 h). 1-2 h before the dissection, remove the solution using a pipette. Wash once with ddH₂O and dry in the cell culture hood.

4. Processing Tissues for 8 day old Sprague-Dawley Rats.

1. Put a 100 mm dish on ice. Decapitate the rat pups into the dish using a pair of sterilized scissors.
2. Insert the scissors into the foramen magnum and cut the two sides of skull, from the ears to the eyes. Lift the skull using a pair of forceps. Ensure that the whole brain is in the skull. Isolate the cerebellum and put it into the abovementioned 35 mm dish using a pair of forceps.
3. Work under a dissecting microscope. Remove the meninges and blood vessels with two pairs of forceps.
4. Chop the tissues with a blade. Put the tissues into tube 1. Centrifuge for 5 min at RT and 1,500 x g.
5. Aspirate the supernatant. Save the pellet.
6. Add the solution in tube 2 to the pellet. Resuspend by shaking gently.
7. Place the tube in a 37 °C water bath for 15 min. Gently shake every 3 min.
8. Add the solution in tube 4 to this solution. Shake and centrifuge for 5 min at RT and 1,500 x g.
9. Aspirate the supernatant. Save the pellet.
10. Add the solution in tube 3 to resuspend the pellet.
11. Prepare two 15 mL tubes. Place half of the cells in each tube. Pipette up and down 60-70x using a cotton-plugged Pasteur pipette to homogenize the cells.
12. Add 3 mL of the solution in tube 5 to each 15-mL tube. Let them sit at RT for 10 min and then carefully remove the supernatant with a cotton-plugged Pasteur pipette. Place the supernatant into new 15 mL tubes and centrifuge for 5 min at RT and 1,500 x g.
13. Aspirate the supernatant. Save the pellet.
14. Add culture medium into the pellet to get a cell density of around 1.5 x 10⁶ cells/mL (about 100 mL for 10 pups).
15. Put the cells into culture plates and culture them in the incubator at 37 °C and 5% CO₂.

5. Addition of Ara-C and D-glucose during Culturing

1. After 24 h, add Ara-C stock solution (10 µL/well for 12-well cell culture plates or 20 µL/well for 6-well cell culture plates; final concentration: 1 mM) to inhibit the growth of the glial cells.
2. On day 7, add 50 µL of D-glucose stock solution per well for 12-well cell culture plates or 100 µL per well for 6-well cell culture plates so that final concentration is 1 mM.

6. FDA-PI Double Staining and FDA-PI-Hoechst Triple Staining in CGN Culture

1. Prepare 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 PBS. Mix by vortexing and place this on ice.
   1. For the FDA-PI-Hoechst working solution, add 20 µL of FDA stock solution (final concentration: 10 µg/mL), 50 µL of PI stock solution (final concentration: 50 µg/mL), and 10 µL of Hoechst stock solution (final concentration: 5 µg/mL) in 10 mL of PBS. Mix by vortexing and place this on ice.
      NOTE: Freshly prepare FDA-PI working solution and FDA-PI-Hoechst 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 or FDA-PI-Hoechst working solution (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 or FDA-PI-Hoechst working solution 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, PI, and Hoechst at 520, 620, and 460 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 min after FDA-PI or FDA-PI-Hoechst staining. The exposure time is 100-300 ms and the analog gain is 2.8x.

7. 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 a 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 is no FDA and PI double-positive cell in the overlay images.
2. For FDA-PI-Hoechst triple staining, overlay the images of the cells detected after filtering different fluorescence filters by dragging the FDA-positive layer on the PI-positive layer in a graphics editor software. Adjust the opacity of the FDA-positive layer by entering "50%" in the "Opacity" field of the "Layers" panel.
   1. Merge two layers by clicking on the "Merge Visible" button in the "Layer" menu. 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. Merge the two layers by clicking on the "Merge Visible" button in the "Layer" menu.
3. Visually distinguish large, irregular glial cells from CGNs by comparing the images taken under fluorescent mode with those taken under phase contrast mode—cells with diameters three times larger than CGNs are considered glial cells and should be excluded.
4. Count the number of viable neurons and dead neurons, respectively, by using a cell counter plugin in an image processing program (e.g., ImageJ)⁴.
   1. For FDA-PI double staining, manually mark small FDA-positive cells as viable neurons. Manually mark PI-positive cells as dead neurons. For FDA-PI-Hoechst triple staining, manually mark small FDA- and Hoechst-double-positive cells as viable neurons. Manually mark PI- and Hoechst-double-positive cells as dead neurons.
5. Calculate the percentage of neuronal viability by using the following equation: % of neuronal viability = [number of viable neurons / (number of dead neurons + number of viable neurons)] x 100%. For each well, choose five random fields and average the percentage of neuronal viability.

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