Antiviral Activity Assay Using SRB Method

Antiviral activity was analyzed using the SRB method to analyze cytopathic effect (CPE) reduction, as previously reported [18 (link)]. Briefly, A549 cells were seeded in a 96-well plate at a density of 3 × 10⁴ cells per well and incubated for 24 h. Following this, 1 × 10³ pfu/90 µL of virus suspension containing TPCK-trypsin (1 µg/mL) was added to each well, along with the indicated concentration of chrysin. The cells were incubated at 37 °C in 5% CO₂ for 3 days until an appropriate CPE was achieved. After 3 days, the A549 cells were washed with PBS and incubated for 30 min at −20 °C after the addition of ice-cold acetone (70%). After removing the acetone, the plate was dried for 30 min in a drying oven, after which, 0.4% (w/v) SRB in 1% acetic acid solution was added to each well and incubated for 30 min at 20 °C. The SRB solution was then removed and the plates were washed with 1% acetic acid before oven-drying. After drying for 1 day, SRB was solubilized with a 10 mM unbuffered Tris-based solution, and the absorbance was measured at 540 nm using a SpectraMax i3 microplate reader (Molecular Devices, Palo Alto, CA, USA) with a reference absorbance of 620 nm. The antiviral activity of each test compound in A/PR/8 virus-infected cells was calculated as a percentage of the corresponding untreated control.