In this example, samples include virus 1 (calculated pH 5.81), virus 2 (calculated pH 5.35), and virus 3 (control virus, used to estimate the baseline reading). Relative light units (RLUs) are output to csv files, which are then analyzed in GraphPad Prism 8 to calculate the point of inflection by nonlinear regression with a dose-response equation. Luminescence is measured using a plate-reader luminometer. (D) Quantification of reporter gene expression and calculation of virus inactivation pH. After 17-19h incubation at 37☌ and 5% CO 2, media is aspirated, 20 μl of lysis buffer is added, and 100 μl of diluted Renilla luciferase assay substrate is added. A multichannel pipette is used to transfer 200 μl re-neutralized virus to a white 96-well tissue culture (TC) dish containing MDCK-Luc9.1 cells. A multichannel pipette is used to transfer 90 μl of sample from panel A into 810 μl infection media that has a pH of 7.0. 5-μl aliquots of twelve virus or control samples are added to eight wells each and incubated at 37☌ for 1h. Into each well of a 96 deep-well plate, 495 μl of pH-adjusted PBS is added.
Schematic of luciferase reporter assay to measure influenza virus inactivation pH.
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