Technical Resources

Plaque Assay Procedure

This is our preferred protocol which we use routinely at Virapur to perform plaque titration and agarose overlay assays. The agarose overlay should be performed on your initial transfection to enhance the possibility of picking single clonal viruses. If you want to outsource this assay, contact Virapur. Information and pricing can be obtained by calling (858) 824-9000 or email us to arrange this testing. Send our viral experts your samples to be titered and we will generate answers or deliver isolates within 2-3 weeks.

Definitions
Materials and Equipment
Collagen Coating Plate Procedure
Harvesting and Plating Cells
Dilution and Infection
Infection of Monolayers
Agar Overlay
Plaque Visualization
PFU per mL calculations

DEFINITIONS:

• Plaque: an area of cells in a monolayer which display a cytopathic effect, e.g. appearing round and darker than other cells under the microscope, or as white spots when visualized by eye; the plaque center may lack cells due to virus-induced lysis.
• Plaque-forming unit (PFU): a virus or group of viruses which cause a plaque.

Note: Procedure provided by Dr. Atsushi Miyanohara and Kathy Bouic of the Gene Thaerapy Program at the University of California, San Diego.

MATERIALS AND EQUIPMENT

• Cell Growth Media (DME high glucose with 4 to 6 mM glutamine and 10% fetal bovine serum) Pen-Strep and Fungizone can be added if desired.
• Plaquing Medium (DME high glucose with 4 to 6 mM glutamine and 2% fetal bovine serum)
• PBS, no Ca or Mg
• Agarose, UltraPure (Gibco Cat. No. 15510-019 or equivalent)
• Tissue culture grade water (WFI)
• Eppendorf or similar pipettors capable of 10 to 100 microliter dispensing
• Tubes to make your dilutions
• 65ºC water bath
• Microwave oven
• Vortex
• Various tissue culture grade sterile bottles of appropriate size (100-250 ml)
• MTT (Sigma, M2128)
• 6 well tissue culture dishes from Corning pre-coated by YOU with a solution of Collagen (Celltrix, Vitrogen 100) OR
• 6 Well Tissue culture dishes pre-coated with Collagen are commercially available from Biocoat, Falcon
• Collagen precoated surfaces enhance the ability of the monolayer to remain attached to the dish during manipulations. Collagen treated dished are not absolutely necessary, but give you an extra advantage in helping the cells adhere to the plastic during manipulations.

COLLAGEN COATING PLATE PROCEDURE

1. Prepare a solution of 0.5 mg/mL collagen (Celltrix, Vitrogen 100 ) in 1 mM acetic acid.
2. Prepare 6 mL of this solution per six-well plate to be used. Filter the solution through a 0.22 micron filter.
3. Add 1.0 mL of collagen solution to each well. Incubate for at least 30 minutes at RT.
4. Using good sterile technique, pipet off and save the collagen solution for future use. You can use the plate(s) immediately or you can let the plate(s) dry covered in a sterile environment overnight. When dry, recover the plates and use immediately or reseal in plastic bags and store at room temperature until use.

HARVEST AND PLATING OF 293 CELLS IN 6-WELL DISHES

1. Make a solution of cells at 1 x 10⁶ cells per mL in Growth Media
2. Add 2.0 mL of growth media with cells to each well. Shake the plate to evenly distribute cells, 12 o’clock to 6 o’clock and 9 o’clock to 3 o’clock (left right top bottom). Incubate plates in a 37ºC, CO₂ incubator.
3. In ~12-24 hours, the cells should be ~90-100% confluent. Go on to the next step immediately. Waiting several more days for cells to become completely confluent is not optimal.

Ad5 DILUTION AND INFECTION

1. When using unknown concentrations of virus directly from infected cell culture, supernatant plus lysed cells should exhibit a titer of 10⁸ through 10⁹ PFU per mL. Optimal levels of purified virus will have a titer from 10⁹ through 10¹⁰ PFU/mL.
2. Prepare dilutions of virus in PBS. Never make more than a 100 fold dilution; always pipette at least 10 µl of virus or solution to reduce pipetting error.
   How we usually dilute virus at Virapur:
3. Prepare 4 tubes; each tube will contain 2 mL of PBS
4. Add 20 microliters of virus sample to the first tube. Vortex.
5. Repeat the dilution process through all four tubes.
6. The tubes will now have these effective dilutions of virus: -10²(1/100), -10⁴ (1/10,000), -10⁶ (1/1,000,000), -10⁸ (1/100,000,000).

INFECTION OF THE MONOLAYERS

1. Pipet off and discard ONE ML of media from each well. One mL of media should now remain on each monolayer.
2. Add 100 µL or 10 µL of each dilution in duplicate to each well, letting the virus flow gently into the media.
   [Consider this: If 100µL of a 10⁶ dilution yields 25 plaques, the titer of virus is 2.5x10⁸ PFU/mL. If 10uL of a 10⁶ dilution yields 30 plaques, the titer of virus is 3.0 x10⁸ PFU/mL.]
3. Incubate the infected monolayers at 37ºC for four to sixteen hours; mildly shake the plates gently several times during this adsorption period.

AGAR OVERLAY

1. Prepare a sterile solution of 4% agarose in dH₂O by autoclaving at 121ºC for 20 minutes. Agarose may be stored on the shelf at room temp or used immediately after equilibrating in a 65ºC water bath. Alternatively, 100 mL aliquots of solid agarose can be melted in a microwave for about 1 minute and cooled to 65ºC in a water bath. You can burn agarose in the autoclave, so dont leave the agarose solution in a hot autoclave overnight.
2. Warm the plaquing media (see above, 2 mL per well to be overlaid) in a 37ºC water bath until equilibrated. Make absolutely sure your media is at 37ºC.
3. Gently draw media out of each Ad5 infected monolayer well and discard. We suggest a pasteur pitpette attached to a vacuum.
4. Mix the volume of media you need ( i.e. 5 plates, 30 wells, 60 mL) to a 37ºC prewarmed container and add 0.11 volumes of liquid agarose to the bottle with swirling (1:10 dilution). Shake vigorously to mix. Do not be afraid to mix well and immediatly.
5. With a new pipette, immediately but gently add 2 mL of the agarose/growth media to each well, pipetting it down the side of the well.
6. Let the plate(s) sit for 15 minutes in the level hood at room temp as the agar overlay turns solid.
7. Move the plate(s) to a humidified incubator at 37ºC and 7.5 to 10% CO₂.

PLAQUE VISUALIZATION

• Plaques will be visible by day 5 to 7 after infection, and if you desire you can stain the monolayer to visualize and count the plaques on the final day of plaque development. With the naked eye, look for white dots on the monolayer. These dots may be more easily visualized by viewing the plate with oblique light. Note: It is critical to confirm that the dots are plaques by inspection under a microscope.
• If desired, stain the plate(s) at 5-6 days post infection or when plaques have fully developed. Stain 3 hr at 37ºC by adding 0.1 volume of MTT solution (5 mg/ml in PBS) (Sigma, M2128). You can pick viable virus plaques through the MTT staining. Sterilize MTT solution by filtration if plaques will be picked. The monolayer will appear blue/black and plaques will be clear areas. This method of staining gives much clearer plaques than neutral red staining.

PFU/mL CALCULATION

• After counting plaques, you can calculate the concentration of the initial viral suspension in PFU/mL.
• Take the dilution of virus you plated and the volume of virus solution you placed on the single monolayer to determine the PFU/mL .
• Example: If you placed 10 µl of a –10⁶ dilution on a well, you will see 44 plaques. The titer from that well is 4.4 x 10⁹ PFU/mL; your initial virus suspension is 4.4 x 10⁹ PFU/mL.
• Example: If you placed 100 ul of a –10⁸ dilution on a well, you will see 88 plaques. The titer from that well is 8.8 x 10¹⁰ PFU/mL.
• Perform the appropriate statistical calculations if you like. At Virapur, we find that this assay is consistent within about a factor of 2 to 3!