Pierce firefly luciferase glow assay with pierce firefly signal enhancer

HeLa cells were plated at 10⁴ cells/well in 100 µl of medium and incubated overnight at 37°C, 5% CO₂ and 98% humidity. MZC or CG was diluted in medium to obtain 2× dilutions of the appropriate dilution range to test (0.000067 to 0.00000001). The cell culture media was then removed from the microplates and replaced with 50 µl of the dilutions of each formulation or 50–100 µl of medium for virus and cell controls. Dilutions were tested in triplicate. HPV PsV stocks were thawed on ice and diluted to 10⁷ copies/ml. Fifty μl of PsV (5×10⁵ copies) were added to all wells with the exception of cell controls and incubated for 72 h at 37°C, 5% CO₂ and 98% humidity. The cells were lysed to detect luciferase activity using the Pierce Firefly Luciferase Glow Assay with Pierce Firefly Signal Enhancer (Thermo Scientific) as described by the manufacturer. Luminescence was read on a Gemini EM microplate reader (emission 542 nm) using Softmax Pro 3.2.1 software. Cytotoxicity for each gel formulation was estimated using the XTT assay [27] (link), [28] (link). For this purpose, the antiviral assay was mimicked but in the absence of virus. CC₅₀ and IC₅₀ values were calculated using a dose-response-inhibition analysis on GraphPad Prism v5.0c software. Therapeutic indexes (TI = CC₅₀/IC₅₀) were calculated.