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Baculovirus Plaque Assay - Neutral Red Staining

Baculovirus Plaque Assay - Neutral Red Staining
Contributor: The Laboratory of Donald Rio at the University of California, Berkeley
This assay is useful for non-LacZ-expressing recombinant viruses (e.g. pVL1392/1393 derivatives)
1. Seed four 60 mm dishes with 2 million cells/plate per virus stock to be titered. Incubate the plates at 27°C for 1 hr to allow the cells to attach.

2. Dilute the virus stocks 10-3 to 10-7 in log-increments (see Hint #1).

3. Aspirate the media from each 60 mm plate and put 1 ml of the dilutions onto each plate (see Hint #2). Mark the top of the plates.

4. Rock the plates at room temperature for 1 hr.

5. While the plates are rocking, get the media/Agarose ready. Put 45 ml of Complete TNM-FH Media into a 100 ml tissue culture bottle and place in a 50°C water bath. Melt 20 ml of 5% Low-Melt Agarose in a microwave and be sure that the Agarose is fully dissolved (see Hint #3).

6. As the end of the 1 hr of plate rocking approaches, add 5 ml of the molten 5% Low-Melt Agarose to the 45 ml of Complete TNM-FH Media at 50°C to make the overlay solution. Remove the bottle from the 50°C water bath and allow the overlay solution to cool to about 40°C (i.e. until you can comfortably hold the bottle in your bare hand).

7. Aspirate the virus inoculum starting with the 10-7 dilution plates and going down in dilution so you can use the same pipette. Remove all the liquid and any bubbles from the edge of the plate.

8. Gently add 5 ml of the overlay solution down the side of the plate so that the cells are not disturbed. Do not move the plates again until the overlay solution has hardened to a solid.

9. Allow the overlay to harden for 10 to 20 min.

10. Put the plates right-side-up in a zip-lock plastic bag in a 27°C infected-cell incubator for 4 days.

11. After 4 days, prepare a 0.5% Low-Melt Agarose solution containing 200 μg/ml Neutral Red. Melt 50 ml of 0.5% Agarose in a microwave and transfer 20 ml of it to a 50 ml conical tube. Add 0.4 ml of a 10 mg/ml Neutral Red Stock solution. Mix the Agarose/Neutral Red solution and allow it to cool to 37°C to 40°C.

12. Add 2 ml of the Agarose/Neutral Red solution per 60 mm dish. Let the Agarose/Neutral Red solution to harden for 10 to 20 min. Place the plates in a zip-locking plastic bag in the 27°C incubator overnight.

13. Count the plaques that appear as light, whitish areas surrounded by live Neutral Red-staining cells.

14. A good high titer virus stock should be about 1 to 5 X 108 plaque forming units (pfu)/ml. If you have 123 plaques on the 10-6 dilution plate, the stock is 1.23 X 108 pfu/ml.

Neutral Red Stock   10 mg/ml Neutral Red
0.5% Low-Melt Agarose   0.25 g Low-Melt Agarose in 50 ml ddH2O
Mark volume level and autoclave.
After autoclaving, add ddH2O to original volume.
Microwave until Agarose is dissolved.
5% Low-Melt Agarose   Mark volume level and autoclave.
2.5 g Low-Melt Agarose in 50 ml ddH2O
After autoclaving, add ddH2O to original volume.
Microwave until Agarose is dissolved.
TNM-FH Insect Medium   2 mM Glutamine
10% Fetal Calf Serum
100 Units/ml Penicillin
100 μg/ml Streptomycin
TNM-FH Insect Medium (Complete)
BioReagents and Chemicals
Agarose, Low-Melt
Fetal Calf Serum
TNM-FH Insect Medium
Neutral Red
Protocol Hints
1. This is conveniently done in a 5 ml polystyrene tube using 1 ml serum-free TNM-FH Insect Medium. Put 1 μl of the virus stock in the 10-3 tube and mix. Take 100 μl of this stock and add it to the 10-4 tube containing 1 ml of serum-free TNM-FH medium. Repeat this 1:10 dilution pattern to the 10-7 tube. Use a separate pipette tip to make each dilution.

2. If you start with the 10-7 dilution and work up to the 10-3 dilution, you can use the same pipette tip for all the additions.

3. The 5% Agarose is very viscous and needs to be at the correct concentration before adding it to the TNM-FH Media.