Dual-Luciferase Transient Assay Protocol

For the transient assays, 1.0 x 10⁵ cells were transfected using Lipofectamine LTX 2000 (Invitrogen) with 1 μg of each Luciferase construct and 100 ng of pRL-SV40 vector (Promega), according to the manufacturer’s instructions. Firefly and Renilla Luciferase activities were measured in cell lysates 48 hours after transfection using the DualGlo Luciferase Assay System (Promega) on a Veritas TM Microplate Luminometer (Perkin-Elmer) following the manufacturer’s protocol and as shown in our companion paper and as described previously (48 (link), 55 (link)). All experiments were done in triplicate. Ratios of Renilla luciferase readings to firefly luciferase readings were taken for each experiment, and triplicates were averaged. The average values of the tested constructs were normalized to the activity of the empty pGL3-basic vector, which was arbitrarily set at value 1.

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Ghatak S., Hascall V.C., Karamanos N., Markwald R.R, & Misra S. (2022). Interplay Between Chemotherapy-Activated Cancer Associated Fibroblasts and Cancer Initiating Cells Expressing CD44v6 Promotes Colon Cancer Resistance. Frontiers in Oncology, 12, 906415.

Publication 2022

Lipofectamine LTX 2000 pRL-SV40 vector DualGlo Luciferase Assay System Veritas TM Microplate Luminometer

Assay Cell Companion Firefly Firefly luciferase Lipofectamine 2000 Luciferase Pgl3 Promega Renilla luciferase Sv40 Transfection Transient Vector

Corresponding Organization : Medical University of South Carolina

Other organizations : Cleveland Clinic, University of Patras

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Variable analysis

independent variables

Luciferase constructs (1 μg of each)

dependent variables

Firefly Luciferase activity
Renilla Luciferase activity

control variables

Cell number (1.0 x 10^5 cells)
Transfection reagent (Lipofectamine LTX 2000)
Transfection of pRL-SV40 vector (100 ng)
Measurement time (48 hours after transfection)
Measurement method (Dual-Glo Luciferase Assay System)

controls

Positive control: pGL3-basic vector (empty vector)
No negative control specified

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