Culturing Primary and Transformed Neuronal Cells for Studying Pseudorabies Virus Infection

Toh Hean Ch'ng, E. Alexander Flood, and Lynn William Enquist

Summary

This chapter discusses the culture of primary sympathetic neurons (superior cervical ganglia) from rat embryos and PC12 cells differentiated into neurons for use in viral infection experiments. Methods are described for the use of a neurotropic herpesvirus, pseudorabies virus (PRV), to analyze the assembly, egress, and transport of viral antigens in neurons.

Key Words: Cultured neuron; herpesvirus; immunofluorescence; infection; laminin; nerve growth factor; neuron; ornithine; PC12; pheochromocytoma; pseudorabies virus; superior cervical ganglia; sympathetic neurons.

1. Introduction

This chapter focuses on the culture of primary sympathetic neurons (superior cervical ganglia) from rat embryos and PC12 cells differentiated into neurons for use in viral infection experiments. Although methods for culturing these types of neuronal cells are well established (1,2), it is often necessary to modify the cell culture method to suit the requirements of a particular experiment. We use these neuronal cultures to understand how the neurotropic herpesvirus-es traverse the long distances of axons between nerve cells. An important technical advance was to develop a protocol for analyzing infected cultures by immunofluorescence assays. This assay is challenging because the cytopathic effects of viral replication weaken the attachment of the neurons to their substrate, and the neurons are easily washed away during changes of medium. The following methods were developed for use of the neurotropic herpesvirus pseudorabies virus (PRV) to analyze the assembly, egress, and transport of viral antigens in neurons (3). PRV is a useful model to study herpesvirus infection because it is closely related to the human herpesviruses, herpes simplex and

From: Methods in Molecular Biology, vol. 292: DNA Viruses: Methods and Protocols Edited by: P. M. Lieberman © Humana Press Inc., Totowa, NJ

varicella-zoster. Additionally, PRV infects a wide range of mammals, except for higher primates, providing flexibility in cell culture use and relative safety for laboratory workers.

2. Materials

2.1. Preparation of Serum-Free Culture Media and Culture Dishes

2.1.1. Serum-Free Neuronal Culture Media

1. IX F12 nutrient mixture (Ham's) liquid (Invitrogen).

2. 1X Dulbecco's modified Eagle's medium (DMEM), liquid, high glucose (Cellgro).

3. Insulin, bovine pancreas, powder (Sigma). Dissolve insulin in tissue culture-grade water (Sigma), and filter-sterilize. Working solution is stable at 4°C for 1 wk.

4. Human holo-transferrin, powder, cell culture grade (Sigma). Dissolve transferrin in 0.9% saline solution, filter-sterilize, and store in -20°C freezer. Do not freeze-thaw. Keep thawed aliquots at 4°C. Discard after 2 wk.

5. Putrescine (tetramethylenediamine dihydrochloride; Sigma). Dissolve putrescine in 0.9% saline solution, filter-sterilize, and store in -20°C freezer. Do not freeze-thaw. Keep thawed aliquot at 4°C. Discard after 2 wk.

6. Progesterone, cell culture (Sigma). Dissolve progesterone in tissue culture-grade water, filter-sterilize, and store in -20°C freezer. Do not freeze-thaw. Keep thawed aliquot at 4°C. Discard working solution after 1 mo.

7. Selenium (in sodium selenite form; Sigma). Dissolve sodium selenite in tissue culture grade water, filter-sterilize, and store stock at -20°C freezer. Prepare and store working solution in 4°C. Discard stock solution after 1 yr.

8. Glucose. Dissolve glucose in tissue culture-grade water, filter-sterilize, and store at 4°C.

9. 200 mM l-Glutamine (Invitrogen). Keep frozen aliquots of l-glutamine at -80°C. Discard remainder of aliquot after use.

10. Bovine serum albumin (BSA), fraction V via cold alcohol precipitation (Sigma).

11. Penicillin/streptomycin, liquid (Invitrogen).

12. Nerve growth factor, 2.5S (Invitrogen; see Note 1).

13. Luer-Lok sterile syringe with different volumes (Becton Dickinson).

14. 0.22-|im Syringe-driven filter units (Millipore).

15. 50-mL Disposable screw-cap conical tubes (Sarstedt).

2.1.2. Preparing Neuron Culture Dishes

1. Poly-dl-ornithine hydrobromide 3000-15,000 MW (Sigma). Dissolve poly-dl-ornithine in 0.1 M borate buffer, pH 8.3.

2. Polystyrene tissue culture dishes (35 x 10 mm; Becton Dickinson).

3. Natural mouse laminin (Invitrogen).

4. Saline G (Sal G) buffer, pH 7.4: 6.1 mM glucose, 5.3 mM KCl, 136.9 mM NaCl, 1.1 mM KH2PO4, 1.08 mM Na2HPO4, 0.11 mM CaCl2 (anhydrous), 0.63 mM MgSO4 (see Note 2).

5. Ca2+/Mg2+-free (CMF) saline G buffer, pH 7.4: 6.1 mM glucose, 5.3 mM KCl, 136.9 mM NaCl, 1.1 mM KH2PO4, 1.08 mM Na2HPO4 (see Note 2).

6. Tissue culture-grade water (Sigma).

2.2. Culturing Dissociated Rat Embryonic Sympathetic Ganglia

2.2.1. Dissection of Embryonic Rat Sympathetic Ganglia

1. 6-in Tissue forceps (1 x 2 teeth; Roboz Surgical).

2. 5.5-in Straight, sharp-blunt operating scissors (Roboz Surgical).

3. Dumostar no. 5 microdissecting tweezers (Roboz Surgical).

4. Two 125-mm no. 2 microdissecting knife (blade 1.7 x 27 mm; Roboz Surgical).

5. Two 100-mm Pyrex glass Petri dishes bottoms (Corning).

6. Two 100-mm bacterial polystyrene Petri dishes (Fisher).

7. Saline G buffer.

8. Glass Pasteur pipets with flame-smoothened tips.

9. Styrofoam surgical stage layered with dissection diapers.

10. Pregnant Sprague-Dawley female rat at stage E15.5 (Hilltop).

2.2.2. Plating the Dissociated Sympathetic Neuron Culture

1. CMF saline G buffer (see Subheading 2.1.2., item 5).

2. Glass Pasteur pipets with flame-smoothened tips.

3. Poly-dl-ornithine and laminin-treated tissue culture dishes.

4. Trypsin (Worthington Biochemicals). Dissolve trypsin in CMF saline G buffer, and filter-sterilize.

5. Trypsin inhibitor, type I-S from soybean (Sigma). Dissolve trypsin inhibitor in CMF saline G buffer, and filter-sterilize.

6. Serum-free neuronal culture media.

2.2.3. Maintaining the Dissociated Sympathetic Neuron Culture

1. Serum-free neuronal culture media.

2. Cytosine P-d-arabinofuranoside (Ara-C; Sigma).

2.3. PC12 Cell Culture and Differentiation

1. Tissue culture dishware (Becton Dickinson, Falcon).

3. Horse serum (Invitrogen).

4. Fetal calf serum (FCS; Invitrogen).

5. Nerve growth factor 2.5S subunit, murine (Invitrogen; see Note 1).

6. Growth medium: 85% RPMI, 10% horse serum, 5% FCS.

7. Differentiation medium: 99% RPMI, 1% horse serum, (50 pg/mL) nerve growth factor (NGF).

8. Freezing medium: 80% RPMI, 10% horse serum, 10% dimethylsulfitide (DMSO).

9. 0.1 M Borate buffer, pH 8.3, filter-sterilized.

10. 10X HEPES-buffered saline solution (HBSS): 1.3 M NaCl, 100 mM HEPES, 54 mM KCl, 42 mM NaHCO3, 4.5 mM KH2PO4, 3.4 mM Na2HPO4 Adjust pH to 7.2 with NaOH, filter-sterilize, and store 45-mL aliquots at -20°C.

11. Natural mouse laminin. Store 20-|jL aliquots at -80°C. (Invitrogen).

12. Poly-dl-ornithine hydrobromide 3000-15,000 MW (Sigma).

13. Rat tail collagen, type 1. Store at 4°C, up to 3 mo (Becton Dickinson Biosciences).

2.4. Infection and Immunofluorescence of Neuron Cultures

1. High titer viral stock.

2. 1X DMEM, liquid, high glucose, (Cellgro).

3. 16% Paraformaldehyde ampules (Electron Microscopy Sciences).

4. 3% BSA (Roche) in phosphate-buffered saline (PBS; Cellgro).

6. Infection medium: 99% RPMI, 1% horse serum.

7. Appropriate primary and secondary antibodies.

8. Vectashield mounting medium for fluorescence samples (Vector Laboratories).

10. Nail varnish.

11. Aqua Poly/Mount (Polysciences).

12. 18-mm Round glass no. 1 cover slips (Fisher Scientific, cat. no. 1254584-18cir1D) (see Note 3).

3. Methods

3.1. Preparation of Serum-Free Neuron Culture Media and Culture Dishes

Our laboratory uses a serum-free and chemically defined medium to culture the sympathetic neurons. By using serum-free media, we eliminate the variability arising from different batches of serum. Typically, a single dissection of 10 embryos (20 superior cervical ganglia) can yield sufficient dissociated neurons for twenty 35-mm tissue culture dishes. The density of each culture can vary depending on the number of ganglia dissected or the number of tissue culture dishes used for plating. Since each dish requires 2 mL of neuron culture medium, prepare 40 mL of the medium prior to dissection. To avoid any contamination, it is important to filter-sterilize each individual stock solution.

3.1.1. Serum-Free Neuronal Culture Media

1. Pipet 10 mL of Ham's F12 nutrient mix into a 50-mL disposable conical tube.

2. Add 400 mg of BSA into the tube, and mix well by vigorous pipeting. Bring volume up to 40 mL by adding 10 mL of Ham's F12 and 20 mL of DMEM.

3. Filter-sterilize basal medium using a 0.22-^m syringe-driven filter unit.

4. Add each supplement listed in Table 1 to the basal medium (see note 4). Mix well.

5. Maintain temperature and pH of the serum-free medium by placing in a 37°C, CO2 tissue culture incubator (see Note 5).

Table 1

List of Supplements in a Serum-Free Neuronal Culture Media

Table 1

List of Supplements in a Serum-Free Neuronal Culture Media

Supplement

Stock concentration

Volume (pL)

Final concentration

Glucose

460 mg/mL

0 0

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