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

• Published in: STAR protocols 2023-03, Vol.4 (1), p.101957-101957, Article 101957
• Main Authors: Pennitz, Peter, Goekeri, Cengiz, Trimpert, Jakob, Wyler, Emanuel, Ebenig, Aileen, Weissfuss, Chantal, Mühlebach, Michael D., Witzenrath, Martin, Nouailles, Geraldine
• Format: Article
• Language: English
• Subjects: Cell Biology; Cell isolation; Protocol; Single Cell
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.101957

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 [Display omitted] •Protocol to isolate murine and hamster lungs with and without prior perfusion•Enzymatic digestion and dissociation to obtain highly viable single-cell suspension•Protocol suitable for infected animals in biosafety laboratory conditions 1–3 Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.