Luciferase-expressing G-pseudotyped VSV∗ΔG-fLuc replicon particles were a gift from Dr Gert Zimmer (Institute of Virology and Immunology) and were produced as described in (41 ). 3×104 HEK293T cells were plated in 96-well plates and cotransfected with 0.04 μg of constructs of interest together with 0.015 μg of a Renilla luciferase-encoding pCAGGS plasmid (pCAGGS-Renilla). The following day, G-pseudotyped VSV∗ΔG-fLuc replicon particle stock was diluted in complete DMEM and cells were infected at an MOI of 0.5. Sixteen hours post-infection, cells were frozen dry at −80 °C for 30 min and then lysed in Passive Lysis Buffer (Promega) for 30 min. Bioluminescence was measured using the Dual-Luciferase Reporter Assay System (Promega) on a microplate reader (Tecan Infinite Lumi). Firefly signals were normalized by Renilla luciferase levels and results were analyzed using GraphPad PRISM.
Infinite lumi
Manufactured by Tecan
Sourced in Switzerland
The Infinite Lumi is a high-performance luminometer designed for sensitive and reliable detection of luminescent signals. It offers a wide dynamic range and fast measurement capabilities, making it suitable for a variety of luminescence-based assays.
Automatically generated - may contain errors
3 protocols using infinite lumi
1
Pseudotyped VSV∆G-fLuc Replicon Assay
2
Cell Viability Assay with CellTiter-Glo 3D
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The CellTiter-Glo 3D Viability-Assay (Promega, Southampton, UK) was mixed with media at a ratio of 1:1 and then incubated 5 min on a plate shaker and 25 min on the benchtop with light protection. Measurements were taken in triplicate using the Tecan Infinite Lumi plate reader (Männedorf, Switzerland). All values were normalised to media control readings.
3
EGF-EGFR Binding Assay
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EGF proteins were coated on the plate wells with coating buffer at 4 °C overnight. EGFR containing His‐tag bound to EGF in a dose ladder (10‐3, 10–2, 10–1, 1, 10, 100 µm ) with PEEPA‐P5 or Vehicle at 37 °C for 2 h and then incubated with His antibody containing horseradish peroxidase (HRP) at 37 °C for 1 h. HRP substrate Tetramethylbenzidine (TMB) was administrated into samples at 37 °C for 20 min and the OD value of each sample was measured by in Infinite Lumi (Tecan).
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