Assay development for live IL-1B detection from individual THP-1 cells

Abstract

Protein secretion is an important process that allows for cell-cell communication and coordinated immune responses to be carried out by immune cells. Methods for the study of cytokine secretion of large cell populations have been well developed, however few methods exist to study cytokine secretion with single-cell resolution. Methods that have been developed, including mRNA assays, flow cytometry with labeled cytokines, ELISPOT assays, and microwell entrapment, have been used to study single-cell cytokine release, but each have drawbacks. One of the main drawbacks is the inability to combine cytokine detection with high resolution imagery of the secreting cells. Such a method would allow the study of intracellular structures and events in combination with cytokine secretion due to external stimuli. To address this, a system was developed that utilizes SU-8 nanopillar structures to suspend cells above a surface, enabling a modified sandwich ELISA to take place underneath individual cells. High-resolution confocal microscopy was used for cytokine detection and simultaneous cellular imaging. Although further optimization must be carried out for a fully functional system to be demonstrated, this project describes the successful development of an antibody coating for the fluorescent detection of IL-1B, and has made steps toward being able to perform live IL-1B secretion studies from THP-1 ASC GFP cells.