ABSTRACT
The TNF superfamily of proinflammatory and proapoptotic cytokines influence tissue-wide responses to molecular insults such as small molecules, toxins, and viral infections that perturb cellular homeostasis at the level of DNA replication, transcription, and translation. In the context of acute lung injury, for example, TNF superfamily members like TNF-α and TRAIL can severely exacerbate disease pathophysiology. This chapter describes a systematic approach to optimization of mammalian cell viability assays and transcriptional profiling through nCounter® Technology to permit a detailed examination of how TNF-α and TRAIL modulate programmed cell death pathways in concert with ricin toxin, a ribosome-inactivating protein (RIP) and a potent inducer of acute respiratory distress. We compare two widely used luciferase- and colorimetric-based cell viability assays and provide optimization protocols for adherent and non-adherent cell lines. We provide a computational workflow to facilitate downstream analysis of datasets generated from nCounter® gene expression panels. While combined treatment with ricin toxin and TRAIL serves as the exemplar, the methodologies are applicable to any TNF superfamily member in combination with any biological agent of interest.