Protocol for laser-induced plasma membrane and cell wall damage in budding yeast

Budding yeast is a model organism to study evolutionarily conserved biological processes, including plasma membrane (PM) repair. Here, we present a protocol to induce localized PM and cell wall damage during live-cell imaging. We describe steps for revival from frozen stock, liquid culture, inducing laser damage, and live-cell imaging. We then detail procedures for fluorescence-signal quantification and statistical analysis. This protocol enables the study of localized PM and cell wall repair mechanisms in budding yeast.