Protocol for high-throughput single-cell patterning using a reusable ultrathin metal microstencil

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 micro -stencil (UTmS). We describe UTmS-chip design, fabrication, and quality charac-terization. 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 applica-tions 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