Optimization of assay parameters.

<p>(<b>A</b>) <b>Determination of RD cell density.</b> RD cells (5×10⁴ cells per well) were incubated in a 96-well plate for 12 h with various dilutions of pseudovirus (from 2×10³ to 2×10⁷ copies per well) in a final volume of 200 μl per well. The data showed that the RLUs decreased with pseudotype in a dose dependent manner. The variation coefficient was lower than 5% for 5×10⁴ and 1×10⁵ cells per well at all the doses of pseudovirus. However, when the RD cell density was 2×10⁴ cells per well, the variation coefficient of RLU was higher than 15% with lower pseudovirus dosage. So the 5×10⁴ cells per well was chosen as the optimized RD cell density. (<b>B</b>) <b>Evaluation of luciferase activity at various incubation times.</b> RD cells (5×10⁴ cells per well) were incubated with 10⁶ copies pseudovirus per well in a 96-well plate for various time points, from 0.5 to 24 h, and each luciferase reading shown is the average of eight replicates. The results indicated that luciferase activity increased gradually with the extension of the incubation over time and reached a plateau at 12 h after incubation. therefore, 12 h was chosen as the optimized incubation time. (<b>C</b>) <b>Infection curve of EV71(FY)-Luc.</b> RD cells were incubated at 35°C together with a range of EV71 pseudovirus concentrations in a final volume of 200 μl per well in 96-well plates. Luciferase activity was measured 12 h post-infection. Each data point represents the average of eight replicates. Linear regression analysis was conducted using Graph Pad Prism. The results showed that when the dose of pseudovirus were within the range of 1×10⁴–10⁷ copies per well, there was a strong correlation between the viral dose and luciferase activity.