- Protocols
- Articles and Issues
- For Authors
- About
- Become a Reviewer
Animal model Antibody analysis Complement analysis
Immune cell differentiation Immune cell function Immune cell imaging Immune cell isolation Immune cell staining Immune mechanisms Immunotherapy Inflammatory disorder Mucosal immunologyAn Ex Vivo Lung Histoculture Model for Studying Pulmonary Infection and Immune Response With SARS-CoV-2 as an Example of RNA Virus
The tissue explant culture (histoculture) is a method that involves maintaining small pieces taken from an organ ex vivo or post mortem in a controlled laboratory setting. Such a technique has a number of advantages: unlike the 2D, organoid, or on-chip cultures, tissue explants preserve the whole complexity of the original tissue in vivo, its structure, extracellular matrix, and the diverse cell populations, including resident immune cells. The explant culture method can be applied to human tissue specimens obtained from biopsies or autopsies, provided that proper ethical protocols are followed. This avoids the difficulties that may arise in translating results obtained on animal models into biomedical research for humans. This advantage makes histocultures especially desirable for studying human pathogenesis in the course of infectious diseases. The disadvantage of the method is the limited lifespan of the cultured tissues; however, a number of approaches allow extending tissue viability to a period sufficient for observing the infection onset and development. Here, we provide a protocol for lung explant maintenance that allows tracing the local effects of infection with SARS-CoV-2 in humans. Further applications of the lung tissues cultured according to this protocol include, but are not limited to, histochemical and immunohistochemical studies and microscopy, FACS, qPCR, and ELISA-based analysis of the conditioned culture media.
VAR2CSA Ectodomain Labeling in Plasmodium falciparum Infected Red Blood Cells and Analysis via Flow Cytometry
Presentation of the variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (EMP1) at the surface of infected red blood cells (RBCs) underpins the malaria parasite’s pathogenicity. The transport of EMP1 to the RBC surface is facilitated by a parasite-derived trafficking system, in which over 500 parasite proteins are exported into the host cell cytoplasm. To understand how genetic ablation of selected exported proteins affects EMP1 transport, several EMP1 surface presentation assays have been developed, including: 1) trypsinization of surface-exposed EMP1 and analysis by SDS-PAGE and immunoblotting; and 2) infected RBC binding assays, to determine binding efficiency to immobilized ligand under physiological flow conditions. Here, we describe a third EMP1 surface presentation assay, where antibodies to the ectodomain of EMP1 and flow cytometry are used to quantify surface-exposed EMP1 in live cells. The advantages of this assay include higher throughput capacity and data better suited for robust quantitative analysis. This protocol can also be applied to other cellular contexts where an antibody can be developed for the ectodomain of the protein of interest.
Modelling Graft-Versus-Host Disease in Mice Using Human Peripheral Blood Mononuclear Cells
Graft-versus-host disease (GvHD) is a significant complication of allogeneic hematopoietic stem cell transplantation. In order to develop new therapeutic approaches, there is a need to recapitulate GvHD effects in pre-clinical, in vivo systems, such as mouse and humanized mouse models. In humanized mouse models of GvHD, mice are reconstituted with human immune cells, which become activated by xenogeneic (xeno) stimuli, causing a multi-system disorder known as xenoGvHD. Testing the ability of new therapies to prevent or delay the development of xenoGvHD is often used as pre-clinical, proof-of-concept data, creating the need for standardized methodology to induce, monitor, and report xenoGvHD. Here, we describe detailed methods for how to induce xenoGvHD by injecting human peripheral blood mononuclear cells into immunodeficient NOD SCID gamma mice. We provide comprehensive details on methods for human T cell preparation and injection, mouse monitoring, data collection, interpretation, and reporting. Additionally, we provide an example of the potential utility of the xenoGvHD model to assess the biological activity of a regulatory T-cell therapy. Use of this protocol will allow better standardization of this model and comparison of datasets across different studies.
Graphical abstract
Induction of Acute or Disseminating Bacterial Pneumonia in Mice and Sampling of Infected Organs for Studying the Host Response to Bacterial Pneumonia
Experimental pneumonia models are important tools to study the pathophysiology of lung inflammation caused by microbial infections and the efficacy of (novel) drugs. We have applied a murine model of pneumonia induced by Pseudomonas (P.) aeruginosa infection to study acute host antibacterial defense in lungs, and assess epithelial cell specific responses as well as leukocyte recruitment to the alveolar space. To study host responses during disseminating pneumonia, we also applied a model of infecting mice with hypermucoviscous Klebsiella (K.) pneumoniae. In the latter model, K. pneumoniae is restricted to lung during the early phase of infection and at the later time points disseminates to the circulation and distal organs resulting in sepsis. Detailed procedures for induction of pneumonia in mice by Pseudomonas and Klebsiella and for isolation and analysis of infected organs, bronchoalveolar fluid, and bronchial brushes are provided in this article.
Phytophthora infestans (Late blight) Infection Assay in a Detached Leaf of Potato
Phytophthora infestans is a hemibiotroph oomycete that primarily infects potato and tomato. It infects stems, leaves, and tubers and fruits of potato and tomato. High throughput and reproducible infection assays are prerequisites to find sources of resistance in any crop. In this protocol, we describe a detached leaf assay (DLA) for conducting the virulence assay of P. infestans in potato leaves. A late blight infection assay using a potato detached leaf is a semi-high throughput assay in which hundreds of plants can be screened in a laboratory setting.
Generating Three-dimensional Human Granulomas in vitro to Study Mycobacterium tuberculosis-host Interaction
Intestinal Enteroid Culture for Human Astroviruses
Bacterial Lawn Avoidance and Bacterial Two Choice Preference Assays in Caenorhabditis elegans
HIV-CRISPR: A CRISPR/Cas9 Screening Method to Identify Genes Affecting HIV Replication
Zebrafish Bacterial Infection Assay to Study Host-Pathogen Interactions
- 1
- 2
- 3
- 4
- 5