387635 High-Throughput Analysis of Single T Cell Calcium Dynamics and Functional Response
T cells are a crucial component of the adaptive immune system, and analyzing T cell response to stimulation is important to better understand functional response to infection. Moreover, cell-to-cell functional variability requires tools that allow single-cell resolution analysis. Current techniques used in analyzing T cell function such as flow cytometry require a large number of cells and lack temporal resolution at single cell level, and therefore masks insightful individual cell dynamic behavior. In addition, ability to track a single cell through time would enable correlation of early time events such as changes in calcium with later time events. Here, we have devised a microfluidic-based platform for stimulating T cells on surface-anchored molecules, while simultaneously analyzing calcium response, and subsequent characterization of cytokine secretion and surface marker expression. We employ established soft lithography techniques in the device fabrication and protein printing techniques to functionalize the surfaces with the specific proteins of interest.
Our platform enables the handling of suspension cells in spatially-defined positions, allowing analysis of hundreds of cells simultaneously at single-cell resolution on a single chip. The platform keeps the cells in place such that they can be tracked through time for dynamic analysis. This enables us to perform later time point assays for late time activation markers and cytokine expression. We show the activation of Jurkat immune cells, and simultaneous fluorescence imaging to track changes in cytosolic calcium. Subsequently, we measure levels of cytokine secretion (IL-2) in the same cells, allowing us to quantify functionally responsive cells. This platform will enable high-throughput analysis of immune cells for rapid screening of immune responsive cells to different stimulants. Overall, our device holds about 4 000 cells on a 2 mm2 footprint, results in 100-fold reduction of reagent use, is compatible with the conventional microscopy imaging techniques, and will enable rapid immunophenotyping of immune cells.