Effector CAR T cells were cocultured with luciferase-expressing target cells at different effector/target (E/T) ratios for 20 h. At the end of the coculture period, supernatant was saved for cytokine assays of IFN-γ, TNF-α, or IL2 levels (R&D Systems). The remaining tumor cells were lysed for 10 min. The luciferase activity in the lysates was measured using the Steady Glo luciferase assay system on Victor (PerkinElmer). Results were analyzed as percent killing based on luciferase activity in wells with tumor cells alone: % killing = 100 − (relative light units [RLU] from wells with effector and target cells)/(average RLU from wells with target cells) × 100.
Steady glo luciferase assay system
Manufactured by PerkinElmer
The Steady-Glo Luciferase Assay System is a bioluminescent reagent used to detect and quantify luciferase reporter gene expression. It provides a stable luminescent signal for continuous monitoring of luciferase activity in cell-based assays.
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2 protocols using steady glo luciferase assay system
1
Effector CAR T Cell Cytotoxicity Assay
2
In Vitro Translation Inhibition Assay
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In vitro toxicity of DON and sulfate derivatives was examined using a commercial in vitro transcription/translation system (TnT® T7 Coupled Wheat Germ Extract System; Promega, Madison, WI, USA). Standard transcription/translation reactions were performed in a total volume of 15 μL according to the manufacturer’s instructions in the presence of the respective compounds in 0.4 % methanol (final concentration). Ribosomes were first preincubated at 30 °C with inhibitors, buffer, amino acids, and DNA. After 7 min, T7-RNA polymerase was added to start the coupled in vitro transcription/translation reactions, which were stopped after 30 min by adding 1 μL of a 1 mM cycloheximide solution. Efficiency of translation was determined by measuring the activity of the firefly luciferase reporter using the Promega Steady-Glo® Luciferase Assay System and the EnSpire® 2300 Multimode Plate Reader from PerkinElmer. Three independent assays using individual dilutions were performed for each substance.
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