Protocols

Plaque Assay

The plaque assay can be used to purify a clonal population of virus or to determine viral titer as plaque-forming units per ml (pfu/ml) so that known amounts of virus can be used to infect cells during subsequent work. In this assay, cell monolayers are infected with a low ratio of virus, such that sporadic cells become infected. An overlay of agarose keeps the cells stable and limits the spread of virus. When each infected cell produces virus and eventually lyses, only the immediately adjacent cells become infected. Each group of infected cells is referred to as a plaque. Uninfected cells surround the plaques. After several infection cycles, the infected cells in the center of the plaques begin to lyse and the peripheral infected cells remain surrounded by uninfected cells. This phenomenon causes the light passing through the infected cells to refract differently than the surrounding uninfected cells, and the plaque can be visualized either by the naked eye or by light microscopy. Each plaque represents a single virus. Therefore, clonal virus populations may be purified by isolating individual plaques. Individual plaques obtained from varying dilutions of a viral stock can be counted to determine the viral titer (pfu/ml) of a given transfection or virus stock. The condition of the cells and their even distribution over the surface of the tissue culture plate is important to the success of a plaque assay. Cells should be healthy, > 95% viable, and in log-phase growth at the time of the assay. Clumpy cells, cells that are not evenly distributed at the correct density (>70%) over the plate, and cells that do not adhere to the tissue culture dishes within 30 min after plating are detrimental to the assay.

Additional Materials Required:

Plaque Assay Agarose, Cat. No. 554766
Grace's Unsupplemented Insect Cell Medium
Fetal Bovine Serum
Tissue Culture Dish, 60 x 15 mm, BD Falcon™ Cat. No. 353802
42°C water bath

Protocol

Determine the number of plates needed

For each co-transfection or virus stock (and positive control) do duplicates of serial dilutions (10^-3, 10^-4, 10^-5, 10^-6). Label each 60 mm plate with sample and dilution descriptions.

Seed culture plate with insect cells

1. Seed 2.3 x 10⁶ Sf9 cells on each 60 mm plate. Gently rock plates back and forth, then side to side to evenly distribute cells on plate surface. Never swirl the plate around; this causes uneven cell distribution. Once plates have been seeded, allow the cells to adhere for 30 minutes to an hour. During this time, prepare agar solution and viral dilutions. Visualize, by light microscopy, to confirm >70% confluency and even cell distribution.

Prepare agarose solution

Plated cells are overlayed with a 1% agarose solution in Grace's Insect Cell Medium supplemented with 10% FBS.

2. First, equilibrate water bath to 42°C. Determine the total volume of agar solution needed: multiply 4 ml/plate by the number of plaque assays. Of this total volume, one half will be autoclaved dH₂O + agarose to make a 2% solution, and the other half will be Grace's Insect Media supplemented with 10% FBS. To determine the amount, in grams, of plaque assay agarose needed to make a final 1% agarose solution, multiply the total volume by 0.01. (For example, 10 plates x 4 ml = 40 ml. 40 ml X 0.01 = 0.4 gm.)
3. Add the appropriate amount of autoclaved dH₂O into an appropriately sized sterile glass bottle. Next, SLOWLY add the calculated amount of agarose, while gently swirling to mix the dH₂O and agarose. Microwave the mixture until just boiling. Remove the bottle and inspect the mixture for any undissolved agarose. If any is present, continue heating. Repeat until the agarose has completely dissolved (CAUTION: mixture and accompanying steam are very hot and can burn. Use appropriate safety gear/precautions). Once agarose has completely dissolved, loosely cap bottle and transfer to the 42°C water bath. Allow mixture to cool to 42°C.
4. Obtain Grace's Insect Media and add Fetal Bovine Serum to 10%. Example: for 100 ml of Grace's Insect Media, add 10 ml of FBS. Place the Grace's media + 10% FBS mix into the 42°C water bath.

Prepare viral serial dilutions

5. For each co-transfection, label six 15 ml sterile tubes from 10^-1 to 10^-6 with the appropriate sample description. Add 2.7 ml of TNM-FH media to each tube. Add 0.3 ml of the co-transfection supernatant to the first tube, vortex tube. Perform serial dilutions by transfering 0.3 ml to the subsequent dilution, using a fresh pipet each time.

Infect monolayer cells

6. At this point, the cells have had ample time to adhere to the plate surface. Check several plates under a light microscope to confirm 70% confluency and even cell distribution.
   
7. Working with the duplicate plates of each dilution, carefully aspirate off media from the cells using a sterile 200 µl pipette tip and vacuum. Do not disrupt the cell sheet. Add 1 ml of the appropriate viral dilution to each of the duplicate plates and gently rock plate back and forth, then side to side to evenly distribute virus. Repeat with all corresponding plates and dilutions.
8. Gently rock plates at room temperature, in sterile hood every 15 minutes for 1 hour.

Overlay infected cells with agarose

9. After 1 hour, confirm the agarose solution is at 42°C. The solution should be warm enough so that it is still in the liquid state, but it should be cool enough to hold by hand. If you cannot comfortably hold the bottle, the solution is too hot and will kill the Sf9 cells. Confirm the Grace's Insect Media w/ 10% FBS is at 42°C. If so, add an equal volume to the agarose solution to complete the final agar solution. Mix the final agar solution well.
10. Once the solution is ready, place in a glass beaker filled with water from the water bath. This should keep the solution from solidifying while you use it under the hood. During the procedure, periodically check the temperature of the water in the beaker. If it is starting to cool, replace it with warm water from the water bath.
11. Working with one duplicate pair at a time, carefully aspirate off media from the cells using a sterile 200 µl pipette tip and vacuum. Do not disrupt the cell sheet. While aspirating, slightly tip the plate and vacuum the supernatant from the edge of the plate. This will allow optimal aspiration without disturbing the cell sheet. Afterwards, slowly add 4 ml of agar solution to each plate and allow agar to solidify.
12. Once the agar on all plates has solidified, carefully place plates in a clean, airtight incubation chamber. Humidify chamber by placing sterile gauze and 10 ml sterile water in a 10 mm culture dish on the bottom rack of the incubation chamber. Seal chamber and place in a 27°C incubator. Allow the plates to incubate for 6 - 10 days.
13. Plaques can be visualized by inverting the plates on a dark background and illuminating them with a strong light source from the side of the plate, or by holding them at a 45° angle into a light source. When learning how to identify a plaque, circle possible plaques with a marker. Using the light microscope, visualize at low power to confirm there is an area of clearing in the lawn of cells. Visualize at high power to look for infected cells at the periphery of the clearing, then less infected cells as you move away from the area of clearing.
14. To facilitate visualization of plaques, overlay with 3 ml of 0.5% agarose (prepared as previously described) containing 50 µg/ml neutral red. (Prepare neutral red (Sigma N7005) stock solution at 1 mg/ml in water or PBS. Filter-sterilize and store 4°C in the dark.) Let harden and incubate plate overnight at 27°C. Neutral red will stain healthy cells and the plaques will appear as clear areas, approximately 0.5 - 3 mm in diameter against a white background. Since neutral red is a vital dye, it is important to stain while the cell monolayer is healthy, ie., 4 to 7 days post infection.

Plaque Purification from Viral Stock

To ensure proper isolation, it is best that plaques are picked from plates containing fewer than 50 plaques. Plate several dilutions of the virus to ensure a sufficiently low number of plaques are obtained. Plaques maybe picked up using sterile micropipette tips (1,000 µl) or microcapillary tubes.

Amplify Virus from Plaque Pickup

1. Mark the plate under the plaque with a marker. Using a sterile pipette tip, remove an agarose plug directly over the plaque. Pick between 10 and 100 plaques in this manner.
2. Place each agarose plug in a separate microcentrifuge tube containing 1 ml tissue culture medium. Elute the virus particles out of the agarose by rotating the tube overnight at 4°C.
3. Add 200 µl of each plaque pickup to separate wells of a 12-well tissue culture plate seeded with 2 x 10⁵ cells per well in 1 ml fresh TNM-FH media. Incubate the plates for 3 days at 27°C.
4. The virus supernatant of this passage-one stock can be collected and centrifuged for 5 min at 1,000 x g at 4°C to remove debris. Store at 4°C.
5. Seed a 100 mm tissue culture plate with 5 x 10⁶ cells for each plaque isolate. Allow cells to attach and replace medium with 10 ml fresh TNM-FH media.
6. Add 200 µl of the passage-one stock to the 100 mm plate and incubate at 27°C for 4 days. Save the remaining 800 µl passage-one stock at 4°C as a backup.
7. Harvest the viral supernatant and centrifuge to remove debris. Determine the titer of this passage-two stock. If the titer remains below 2 x 10⁸ pfu/ml, proceed to Baculovirus Amplification.