Virus aliquots in panels E-H were exposed to pH-adjusted PBS for 1h before inoculation into Luc9.1 reporter cells at an MOI of 2 (E,F) or 0.2 (G,H) PFU/cell. After 17 h, cell lysates were mixed with Renilla luciferase substrate and luminescence (relative light units, RLU) was measured using a 96-well plate luminometer. MDCK Luc9.1 cells were inoculated with A/TN/09 WT (solid circles) or Y17H (open triangles) at MOI values ranging from 2x10 -5 to 2 PFU/cell and were overlaid with media lacking TPCK-treated trypsin (A,B -T) or containing it (C,D +T).
Renilla luciferase assay of residual infectivity after acid exposure using MDCK Luc9.1 reporter cells. Time to conduct assay is after culturing of cells. Total virus is an estimate of total PFUs needed for each assay. (E) Comparison of luciferase and TCID50 assays to measure virus inactivation pH. RLU values were assigned for illustrative purposes only and are not real data.
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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