6. Dishes are now ready for use and may be coated with laminin or stored for 1 wk at 4°C.

3.3.3. Laminin Substrate

Laminin is applied after applying the poly-DL-ornithine. Accordingly, coating glass surfaces with these materials requires two overnight incubations.

1. Thaw laminin on ice to prevent it from gelling.

2. Dilute laminin in 1X HBSS to 10 |Jg/mL.

3. Aseptically coat dishes according to volumes listed in Table 2.

4. Incubate dishes overnight in a humidified incubator at 37°C.

5. Rinse dishes three times with HBSS.

6. Rinse dishes once with differentiation medium.

7. Dishes are ready for use or may be stored for 1 wk at 4°C.

3.4. Preparing Stocks of NGF-Primed PC12 Cells (see Note 18)

1. Coat twelve 15-cm dishes with collagen as described in Subheading 3.3.1.

2. Wash each dish three times with sterile water (~3 mL per dish), and aspirate completely between washes. Rest the dishes at an angle to facilitate collecting the water.

3. Plate undifferentiated PC12 cells into four 15-cm dishes (a 1:3 split ratio), and allow them to grow for 4 d in complete medium.

4. Collect the cells by trituration, and divide them equally among the eight remaining 15-cm dishes. Incubate overnight in growth medium. Cell clumps can be dissociated using either Pasteur pipets or a 23-gage needle. Trypsin cannot be used to dissociate PC12 cells, as even a brief exposure to trypsin is lethal.

5. The next day replace the growth medium with differentiation medium (see Note 19).

6. Replace the differentiation medium every other day for 9 d (total of four changes of medium, exposing cells to NGF for 10 d; see Note 20). After 5 d an extensive network of neurites should be visible (Fig. 4).

7. On the 10th day, collect the cells in a volume of 10 mL freezing medium per 15-cm dish.

8. Freeze 1-mL aliquots in a styrofoam box overnight at -80°C.

Fig. 4. Neurite outgrowth of PC12 cells grown on dishes treated with poly-ornithine and laminin. PC12 cells after 5 d in differentiation medium (A) or in growth medium (B).

9. Move the cells to liquid nitrogen storage.

10. Test differentiation of cells by thawing one aliquot and plating various dilutions of cells in a collagen-coated 24-well plate in differentiation medium. Within 24 h, the primed cells will extend visible neurites (see Note 21).

3.5. Infection and Immuneofluorescence of Dissociated Sympathetic Neurons and NGF-Differentiated PC12 Cells (see Note 22)

Although both sympathetic neurons and PC12 cells attach much better to plastic substrates, some microscopy techniques require that the cells be grown on glass surfaces. The following protocols are routinely used for plating sympathetic neurons on plastic substrates (Subheading 3.5.1.) and differentiated PC12 cells on glass (Subheading 3.5.2.).

3.5.1. Primary Neurons on Plastic Surfaces

1. For a typical PRV infection, prepare the viral inoculum by diluting a high-titer viral stock (1 x 108 PFU/mL) 1:10 with DMEM. Gently remove, and save the neuron culture medium from the dish. Replace the medium with 600 ||L of the viral inoculum. Make sure that the inoculum covers the entire surface of the dish. Place the dish and the neuron culture medium back in the incubator for 1 h. Do not rock or tilt the dish during incubation (see Note 23). Viral particles will attach to and enter the neurons during this 1-h absorption period.

2. After 1 h, remove viral inoculum from the dish, and replace with the original neuron culture medium saved prior to infection. Place the dish back in the 37°C incubator.

3. At the appropriate time after infection, remove dishes from the incubator, and wash each dish twice with PBS. Gently flow buffer down the sides of the dish. After infection, the network of neurons is no longer as firmly attached to the substratum and can be easily dislodged.

4. After the final wash, add 1 mL of 3.2% paraformaldehyde, diluted in PBS. Allow the paraformaldehyde to fix the cells for 10 min before washing the dish three times with PBS.

5. After the final wash, add 1 mL of PBS in 3% BSA to the dish, and store the dish at 4°C, overnight. The BSA in the PBS wash buffer acts as a blocking agent and helps stabilize the cells.

6. The next day, incubate the dish in PBS containing 3% BSA and 1% saponin for 10 min (see Note 24).

7. Remove the buffer, and add 600 |L of the primary antibody diluted in the PBS/BSA/saponin buffer. Incubate for 1 h in a 37°C humidified incubator.

8. Remove the primary antibody, and wash three times with PBS/BSA/saponin buffer. Add 600 | L of the appropriate secondary antibody. Incubate for 1 h as before.

9. Remove the secondary antibody, and wash twice with PBS/BSA/saponin buffer. Rinse the dish one final time with distilled, tissue culture-grade water. Carefully add a drop of munting medium (Vectashield or Aqua Poly/Mount) in the center of the dish. After 30 s, place a cover slip on top of the neuron culture. Or, if cells were grown on cover slips, place a drop of mounting medium on a glass slide, and lay the cover slip on top of the droplet, cell side down.

10. Using a glass Pasteur pipet connected to a vacuum filter, remove excess liquid around the tissue culture dish and around the edges of the coverslip (see Note 25). Be careful not to remove the mounting medium underneath the cover slip.

11. If you use Vectashield, seal the edges of the cover slip with nail varnish. Make sure the nail varnish dries completely before doing any microscopy work. If you use Aqua Poly/Mount, store the dish at 4°C to allow the medium to polymerize overnight (see Note 26).

3.5.2. Primed PC12 Cells on Glass Surfaces

1. Place 18-mm glass cover slips (see Subheading 2.4., item 16) in 12-well tissue culture dishes. Sterilize the cover slip by dipping it in 95% ethanol, and then pass it over an open flame.

2. Coat cover slips with poly-ornithine (see Subheading 3.3.2.), followed by laminin (see Subheading 3.3.3.).

3. Thaw primed PC12 cells, and seed them at a density previously determined empirically to yield cells sparse enough that they are not overlapping, yet dense enough that they provide sufficient samples per field.

4. PC12 cells are infected by gently replacing the culture medium with 500 |L of differentiation medium containing 2 x 105 PFU of PRV. Incubate the cells for 1 h in a cell culture incubator. After the 1-h period of adsorption of virus to the cells, replace the viral inoculum with 1 mL of prewarmed differentiation medium, and return the cells to their incubator.

5. Immunostaining of differentiated PC12 cells is carried out according to steps 3-9 (if using a nonpolymerizing mounting medium, see step 11) in Subheading 3.5.1.

4. Notes

1. It is cost-effective to purify NGF from mouse submaxillary glands (available from Pel-Freez Biologicals). We purify NGF using the protocol described by Mobley et al. (7).

2. Use tissue culture-grade water to prepare buffer solution. Filter-sterilize the buffer with a 0.22-^m filter apparatus.

3. PC12 cells appear to attach more strongly to these cover slips of German origin. Substrate coating is still necessary.

4. It is important to maintain a sterile and contamination-free stock of supplements. Remember to filter-sterilize all the supplements prior to aliquoting the stocks.

5. Loosen the cap on the conical tube to allow proper equilibration of CO2 inside the tube with the incubator. The CO2 buffers the pH of the solution. The levels of CO2 in the incubator should be within the range of 5.5-6.0%, and this level remains unchanged for any subsequent use of the CO2 incubator in this protocol.

6. Place the 35-mm dishes in a larger 15-cm Petri dish to provide a stable secondary container when transporting the dishes.

7. The Sal G buffer used to wash the embryos must be sterile because the wash buffer will be used to coat the flame-smoothened glass Pasteur pipets.

8. It is easier to immobilize the isolated head by inserting one blade in the eye socket. Next, align the other blade at the jaw. Once the blade is fully anchored, remove the first blade from the eye socket, and place it close to the neck at the opposing end of the jaw. The two blades should now be crossed to form an X. Pull the two blades toward each other in one swift stoke to obtain a clean and thin slice of the lower mandible.

9. It is critical to obtain a thin slice of the lower mandible because the thinner the slice, the easier it is to determine the location of the ganglia.

10. Use glass Pasteur pipets with flame-smoothened tips to avoid shearing or lysing the neurons during pipeting.

11. To accelerate the drying process, tilt the 35-mm tissue culture dish slightly by placing the lid at the bottom of the dish to prop it up. Once the dish is dried, the glossy and shiny surface at the bottom of the dish will be replaced by a thin layer of laminin that looks dull and nonreflective.

12. Trituration requires a steady hand. Work fast, but be gentle. Take great care to avoid making too many bubbles in the medium, as this will lower the yield. Do not expect all the ganglia to dissociate fully into neurons. Let the undissociated "chunks" settle to the bottom of the tube.

13. Use the 2-mL wide-bore disposable pipets when adding the serum-free medium to the dish. Set the discharge flow rate of the pipet-aid to "slow," and allow the medium to dribble down the sides of the dish. This procedure should be followed every time any liquid is exchanged to and from the dish. Never aspirate any media from the dish because the neurons are easily dislodged.

14. Older and more mature cultures are able to survive less frequent neuron culture medium replacements.

15. Collagen applied in this manner is not appropriate for glass surfaces. Store collagen at 4°C for up to 3 mo.

16. Do not use less than the amounts listed in Table 2, as smaller dishes require disproportionately larger volumes than the larger dishes. If you notice that the cells are attached to the dish in a ring pattern (i.e., along the outer edge of the dish and relatively sparse in the center), increase the volume of substrate applied and/or the volume of media in which the cells are plated.

17. The d-isomer may allow cells to remain attached for a longer period, since it is not susceptible to host enzymes.

18. Differentiating PC12 cells into neuron-like cells requires 10 d, replacing media every other day. Preparing a large batch at once ensures homogeneity and simpler scheduling demands. We have stored the cells up to a year without noticing any adverse effect.

19. Do not thaw and freeze NGF repeatedly. It is important to store it as small aliquots. We prepare a large amount of RPMI with 1% horse serum and add NGF only to the amount needed for a given day.

20. During differentiation, many cells will detach from the dish. Under these conditions, only the cells that respond to NGF will survive. Presumably the cells not responding to NGF are dying and consequently lift off the dish (8).

21 . When the cells are collected by trituration for storage, the neurites are sheared off. This action does not affect their differentiation. In fact, visibly apparent neurites are not a requirement for PC12 cells to exhibit their neuronal character in response to NGF (9). Additionally, the length of the neurites varies with the cell density: too many cells and neurites will not extend. However, when making stocks of primed PC12 cells, we plate the cells at high density to obtain the most primed cells per unit of surface area and because approximately half of the cells will detach over the 10-d differentiation process. Once primed, differentiated PC12 cells will extend neurites rapidly when replated on collagen-coated or laminin-coated substrates and will retain their neuronal properties (Fig. 5). Upon thawing, wash cells with 20 volume equivalents of RPMI, to remove as much FCS as possible.

22. Approximately 1000-fold fewer plaque-forming units are released from NGF-treat-ed cells vs undifferentiated PC12 cells (data not shown). Furthermore, prolonged growth of PC12 cells with NGF (>21 d) may render the cells refractory to lytic infection (10).

23. The calculated multiplicity of infection (MOI) is around 2000 PFU per cell plated. However, because the virions adhere avidly to the substratum on the surface of the dish and the cell density is quite low, the effective MOI for the infection is much lower and cannot be accurately determined. The least amount of virus needed to infect 95% of all cells should be determined empirically for each virus and/or type of neuron.

24. These protocols can also be adapted for nonpermeabilized, surface staining of neurons. To surface stain omit saponin from all subsequent washes.

25. The tissue culture dish can also be air-dried for 30 min in the dark before sealing the cover slip with nail varnish.

26. The solvents in nail varnish diffuse into the mounting medium and quench the fluorescence along the edges. This is not a problem for samples on a large surface, such

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