Abstract
Exploiting convenient strategies for single-cell preparation while maintaining a high throughput remains challenging. This protocol describes a simple workflow for high-throughput single-cell patterning using a reusable ultrathin metal microstencil (UTmS). We describe UTmS-chip design, fabrication, and quality characterization. We then detail the preparation of flat substrates and chip assembly for single-cell patterning, followed by culturing of cells on a chip. Finally, we describe the evaluation of single-cell patterning and the downstream applications for studying single-cell calcium release and apoptosis. For complete details on the use and execution of this protocol, please refer to Song et al. (2021).1.