Biochemical assays have been long been used to study the cellular output of pleiotropic cytokine TNF-α in bulk, preventing detection of heterogeneity between cells. We propose to overcome this problem by using fluorescent quantum dots to quantify single molecules of TNF-α and subsequently quantify downstream pathways.
TNF-α is a cytokine responsible for a wide range of physiological processes, including tissue regeneration, inflammation, cell survival, and tissue regeneration. This project will focus on the nuclear factor kappa B (NF-kB) downstream pathway of TNF-α. Nuclear factor kappa B is a transcription factor that regulates the expression of more than 500 genes, and when dysfunctional, can be a leading cause of autoimmune and inflammatory diseases. When TNF-α externally stimulates the cell by binding to TNFR-1, NF-kB translocates from the cytoplasm to the nucleus. This translocation continues in an oscillatory fashion between the cytoplasm and nucleus; it is thought that this oscillation is responsible for regulating the expression profiles of genes. To better understand NF-kB gene regulation, we quantified the number of TNF-α bound per cell using quantum dots (QDs) and then detected subsequent cellular output. First, QDs with a CdSe shell and a ZnS core were conjugated to TNF-α bound to biotin. Gel electrophoresis was used to confirm this conjugation. To detect the functionality of the QD-bound TNF-α in initiating NF-kB translocation, fixed cells were imaged using epifluorescence microscopy.