Invitrogen Bac to Bac Expression System

Performing a Viral Plaque Assay, continued
To improve the visualization of plaques, stain the plates using Neutral Red.
Other plaque staining dyes such as Crystalline Blue are not recommended
because they contain organic solvents that kill the host cells. To stain plaques,
you may do one of the following:
• Prepare an agarose solution containing neutral red and overlay this solution
on the plates 4 days post-infection. Count plaques 7-10 days post-infection.
• Prepare a neutral red solution and add to plates for 1-2 hours just prior to
counting plaques (7-10 days post-infection).
Important: If you plan to plaque purify your baculovirus, you should not stain
plaques as neutral red is a known mutagen that can alter your recombinant virus.
Materials Needed
You should have the following materials on hand before beginning:
• Neutral Red (Sigma, Catalog no. N7005)
• Cell-culture grade, distilled water
• Sf-900 II SFM or other appropriate complete growth medium (if preparing
the agarose solution)
• 4% Agarose Gel (if preparing the agarose solution)
• 40ºC water bath (if preparing the agarose solution)
Neutral Red
Staining
Procedure
Preparing a Neutral Red Agarose Overlay (for use on Day 4)
1. Prepare a 1 mg/ml Neutral Red solution in Sf-900 II SFM (or other
appropriate complete growth medium). Filter-sterilize.
2. Combine the reagents below in a 50 ml tube and place in a 40ºC water bath.
1 mg/ml Neutral Red solution 1.5 ml
Sf-900 II SFM
16.5 ml
3. Microwave 4% Agarose Gel until melted, then place in a 40ºC water bath for
5 minutes.
4. Move the 50 ml tube of neutral red solution and the 4% agarose gel to a sterile
hood. Add 6 ml of 4% agarose gel to the neutral red solution.
5. Add 1 ml of the Neutral Red overlay to each well containing plaquing
overlay. Once the agarose has hardened, return plates to a 27ºC humidified
incubator until plaques are ready to count. Plaques will appear as clear spots
on a red monolayer.
Preparing a Neutral Red Stain (for use on Day 7-10 prior to counting plaques)
1. Prepare a 1 mg/ml Neutral Red solution in cell-culture grade, distilled water.
2. Add 0.5 ml of Neutral Red solution to each well containing plaquing overlay.
Incubate for 1 to 2 hours at room temperature.
3. Gently remove excess stain with a pipet or blotter. Count plaques. Plaques
will appear as clear spots in a nearly clear gel against a red background.
continued on next page
37