The traditional plaque assay is a standard method for determining the plaque-forming units (PFU) of respiratory syncytial virus (RSV). However, this method can be time-consuming and expensive due to the small size of the plaques and the need for specific antibodies against the viral fusion protein. To address these challenges, three simplified plaque formation methods have been developed to make the process more efficient and accessible.
**Method One**
1. Seed Hep-2 cells in a 6-well plate at a density of 5×10ⵠcells/ml, adding 3 ml of cell suspension per well.
2. After the cells form a monolayer within 48 hours, remove the growth medium, wash with PBS, and add 400 µl of PBS and 100 µl of diluted virus (10-fold serial dilution) per well.
3. Incubate at 37°C with 5% CO₂ for 1–4 hours, shaking the plate every 15–30 minutes to ensure even distribution of the virus. The optimal adsorption time is typically 4 hours.
4. Discard the adsorption solution, then overlay each well with 3 ml of a mixture made from 1:1 ratio of 2× DMEM containing 5% calf serum and 0.6% agarose. Ensure the final concentrations are 2.5% serum and 0.3% agarose. The agarose must be autoclaved and melted in a microwave before use. Keep the overlay at 65°C until ready to apply.
5. Incubate the plate at 37°C for 6–7 days. Fix the cells by adding approximately 2 ml of 1% formalin (prepared in 0.15 M NaCl) per well and leave it overnight for complete fixation.
6. Remove the overlay and gently rinse any remaining agarose with tap water.
7. Add 2–3 ml of 0.05% neutral red (10× stock solution) to each well. Store the stock solution for several months and dilute as needed.
8. Incubate at room temperature for 1–24 hours, then aspirate the dye and allow the plate to air dry.
9. Count the plaques using standard PFU calculation methods.
**Method Two**
1. Inoculate HeLa cells in a 12-well plate at a density of 5×10²ⵠcells/ml, with 1 ml per well. Allow the cells to form a monolayer within 24 hours.
2. Dilute the virus 10-fold in 10% DMEM (maintenance solution).
3. Aspirate the culture medium, wash the cells with PBS 1–2 times, and add 500 µl of maintenance solution to each well.
4. Add 50 µl of the diluted virus to each well, making two duplicate wells per dilution. Mix well and incubate at 37°C, 5% CO₂ for 2 hours. Shake the plate every 5 minutes to ensure even distribution.
5. Melt sterile 3% agarose in a microwave and keep it at 65°C. Preheat 4% 2× DMEM to 37°C. Mix equal volumes of agarose and DMEM, and add 1 ml of the mixture to each well when the temperature is suitable.
6. Let the plate cool at room temperature or 4°C until the overlay solidifies. Then incubate at 37°C for 4–5 days. Monitor the cells daily and ensure the incubator contains moisture to prevent the agarose from drying out.
7. After incubation, add 100 µl of MTT (0.25% concentration) to each well and incubate for 4 hours. Live cells will stain blue, while plaques remain unstained. Count the plaques and calculate PFU accordingly.
8. The PFU calculation follows the same method as other techniques.
**Method Three**
1. Seed HEP22 cells in a 24-well plate at a density of 2×10ⵠcells one day before the test. Incubate overnight at 37°C, 5% CO₂. Wash the cells with Hanks’ solution 1–2 times, adjust the pH of Eagle’s MEM with NaHCO₃, and dilute RSV 10-fold. Add the diluted virus to the 24-well plate (two duplicates per dilution), and adsorb for 1–2 hours, shaking every 15–30 minutes.
2. Prepare an agar overlay using 2× Eagle’s medium supplemented with streptomycin, kanamycin, sodium bicarbonate, and agarose.
3. After adsorption, discard the virus solution and add 0.5 ml of the agar overlay. Solidify the overlay at 34°C and incubate for 5 days. Fix the cells with formalin and stain with 0.15% crystal violet. Observe under a microscope and count the plaques.
4. Calculate PFU using the formula: PFU = (a × b) / v, where a = average number of plaques, b = inverse of virus dilution, and v = volume of virus added.
These methods offer simpler alternatives to the traditional plaque assay, reducing both time and cost while maintaining accuracy in measuring RSV titer.
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