Kamaishu Biotechnology Analysis Today - Summary of Respiratory Syncytial Virus Plaque Test Methods

The traditional plaque assay is commonly used to determine the PFU (plaque-forming unit) of respiratory syncytial virus (RSV). However, this method can be time-consuming and costly due to the small size of the plaques and the need for specific antibodies against the viral surface fusion protein. To address these challenges, three simplified and more user-friendly plaque formation methods have been developed. These alternatives aim to streamline the process while maintaining accuracy. **Method One** 1. Seed Hep-2 cells into a 6-well plate at a density of 5×10⁵ cells/ml, adding 3 ml of cell suspension per well. 2. After the cells form a monolayer (within 48 hours), remove the culture medium and add 400 µl of PBS along with 100 µl of virus per well (after a 10-fold serial dilution). 3. Incubate the plate at 37°C in a 5% CO₂ environment for 1–4 hours. Shake the plate every 15–30 minutes to ensure even distribution of the virus. The maximum adsorption typically occurs after 4 hours. 4. Discard the adsorption solution and overlay the cells with 3 ml of a semi-solid medium. The overlay is prepared by mixing 1:1 with 2× DMEM containing 5% calf serum and 0.6% agarose, resulting in a final concentration of 2.5% serum and 0.3% agarose. Ensure the agarose is autoclaved, fully melted in a microwave, and kept at 65°C before use. The 2× DMEM should be reconstituted with deionized water, optionally supplemented with antibiotics, filtered, and sterilized before preheating to 37°C. Mix the components carefully to avoid overheating, which could damage the cells. 5. Incubate the plate at 37°C for 6–7 days. Then, add approximately 2 ml of 1% formalin (prepared in 0.15 M NaCl) per well and fix the cells overnight to allow proper penetration into the agar layer. A minimum fixation time of 24 hours is recommended. 6. Remove the agar overlay and gently rinse any remaining agar from the edges of the plate using tap water. 7. Add 2–3 ml of 0.05% neutral red stain to each well. The stock solution is 10× concentrated and can be stored for several months. Dilute it with deionized water before use. 8. Stain the cells at room temperature for 1–24 hours. Afterward, aspirate the dye and gently wash the plate. Allow it to dry at room temperature for future observation. 9. Count the plaques using standard plaque calculation methods. **Method Two** 1. Inoculate HeLa cells in a 12-well plate at a density of 5×10⁵ cells/ml, with 1 ml per well, allowing them to form a monolayer within 24 hours. 2. Dilute the virus with 10% DMEM (maintenance solution) in a 10-fold dilution. 3. Aspirate the growth medium, wash the cells once or twice with PBS, 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. Incubate at 37°C, 5% CO₂ for 2 hours, shaking the plate every 5 minutes to ensure even distribution. 5. Melt sterile 3% agarose in a microwave, then keep it in a 65°C water bath. 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. Allow the plate to solidify at room temperature or 4°C, then incubate at 37°C for 4–5 days. Monitor the cells daily and ensure the incubator has distilled water to prevent the agarose from drying out. 7. After incubation, add 100 µl of MTT solution (0.25% concentration in DDW) to each well. Incubate for 4 hours. Live cells will appear blue, while plaques remain unstained. Count individual plaques to calculate the PFU value. 8. The PFU calculation method is similar to other plaque assays. **Method Three** 1. Seed HEP22 cells in a 24-well plate at a density of 2×10⁵ cells 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 in a 10-fold dilution. Add the diluted virus to the wells (with two replicates per dilution), and allow adsorption for 1–2 hours. Shake the plate every 15–30 minutes to ensure uniform distribution. 2. Prepare an agar overlay using 2× Eagle’s medium supplemented with streptomycin, kanamycin, sodium bicarbonate, and agarose. 3. Remove the virus solution, add 0.5 ml of the agar overlay, and let it solidify at 34°C. Incubate for 5 days, then fix the cells with formalin and stain with 0.15% crystal violet. Observe the plaques under a microscope. 4. Calculate PFU using the formula: PFU = (a × b) / v, where a is the average number of plaques, b is the inverse of the virus dilution factor, and v is the volume of the virus added.

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