| Autor: |
Morris Baumgardt, Maren Hülsemann, Anna Löwa, Diana Fatykhova, Karen Hoffmann, Mirjana Kessler, Maren Mieth, Katharina Hellwig, Benedikt Obermayer, Emanuel Wyler, Simon Dökel, Achim D. Gruber, Ulf Tölch, Stefan Hippenstiel, Andreas C. Hocke, Katja Hönzke |
| Rok vydání: |
2022 |
| Popis: |
Organ models have received widespread attention in the study of SARS-CoV-2, the pathogen causing the current COVID-19 pandemic. Human-based organ models can provide strong predictive value to investigate the tropism, virulence, and replication kinetics of viral pathogens. Applicable to a large set of organoid models and viruses, we provide a step-by-step work instruction for the infection of human lung organoids with SARS-CoV-2 in this protocol collection. We also prepared a detailed description on state-of-the-art methodologies to assess the infection impact and the analysis of relevant host factors in organoids. This protocol collection consists of five different sets of protocols. Set 1 describes the protein extraction from human lung organoids and the determination of protein expression of angiotensin-converting enzyme 2 (ACE2), as an exemplary host factor of SARS-CoV-2. Set 2 provides detailed guidance on the extraction of RNA from human lung organoids and the subsequent RT-qPCR to quantify the expression level of e.g., ACE2or other host factors of SARS-CoV-2 on RNA level. Protocol set 3 contains an in-depth explanation on how to infect human lung organoids with SARS-CoV-2 and how to quantify the viral replication by plaque assay and viral E gene-based RT-qPCR. Set 4 provides a step-by-step protocol for the isolation of single cells from infected human lung organoids for further processing in single-cell RNA sequencing or flow cytometry. Set 5 presents a detailed protocol on how to perform the fixation of human lung organoids and guides through all steps of immunohistochemistry and in situ hybridization to visualize SARS-CoV-2 and its host factors. The infection and all subsequent analytical methods have been successfully validated by biological replications with human lung organoids based on material from different donors. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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