Protocol to dissociate healthy and infected murine- and hamster-derived lung tissue for single-cell transcriptome analysis

Abstract

Summary: In infectious disease research, single-cell RNA sequencing allows dissection of host-pathogen interactions. As a prerequisite, we provide a protocol to transform solid and complex organs such as lungs into representative diverse, viable single-cell suspensions. Our protocol describes performance of vascular perfusion, pneumonectomy, enzymatic digestion, and mechanical dissociation of lung tissue, as well as red blood cell lysis and counting of isolated cells. A challenge remains, however, to further increase the proportion of pulmonary endothelial cells without compromising on viability.For complete details on the use and execution of this protocol, please refer to Nouailles et al. (2021),1 Wyler et al. (2022),2 and Ebenig et al. (2022).3 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Biology
• Cell isolation
• Single Cell