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.

The absence of long term, primary untransformed in vitro models that support hepatitis B virus (HBV) infection and replication have hampered HBV pre-clinical research, which was reflected in the absence of a curative therapy until recently. One of the limitations for in vitro HBV research has been the absence of high titer and pure recombinant HBV stocks, which, as we describe here, can be generated using simple, and reproducible protocols. In addition to infection of more conventional in vitro and in vivo liver model systems, recombinant high titer purified HBV stocks can also be used to efficiently infect differentiated human liver organoids, whose generation, maintenance, and infection is discussed in detail in a companion organoid protocol. Here, we also describe the protocols for the detection of specific viral read-outs, including HBV DNA in the supernatant of the cultures, covalently closed circular DNA (cccDNA) from intracellular DNA preparations, and HBV viral proteins and viral RNA, which can be detected within the cells, demonstrating the presence of a complete viral replication cycle in infected liver organoids. Although an evolving platform, the human liver organoid model system presents great potential as an exciting new tool to study HBV infection and progression to hepatocellular carcinoma (HCC) in primary cells, when combined with the use of high-titer and pure recombinant HBV stock for infection.

Graphical abstract:

The study of host/pathogen interactions at the cellular level during Plasmodium intra-erythrocytic cycle requires differential extraction techniques aiming to analyze the different compartments of the infected cell. Various protocols have been proposed in the literature to study specific compartments and/or membranes in the infected erythrocyte. The task remains delicate despite the use of enzymes or detergents theoretically capable of degrading specific membranes inside the infected cell.

The remit of this protocol is to propose a method to isolate the erythrocyte cytosol and ghosts from the other compartments of the infected cell via a percoll gradient. Also, the lysis of the erythrocyte membrane is done using equinatoxin II, which has proven to be more effective at erythrocyte lysis regardless of the cell infection status, compared to the commonly used streptolysin. The parasitophorous vacuole (PV) content is collected after saponin lysis, before recovering membrane and parasite cytosol proteins by Triton X-100 lysis. The lysates thus obtained are analyzed by Western blot to assess the accuracy of the various extraction steps. This protocol allows the separation of the host compartment from the parasite compartments (PV and parasite), leading to potential studies of host proteins as well as parasite proteins exported to the host cell.

Cytotoxic CD8⁺ T lymphocytes (CTLs) represent a crucial component of the adaptive immune system and play a prominent role in the anti-tumor immune responses of both mice and humans.

Cytotoxic CD8⁺ T cells are responsible for the lysis of cells expressing peptides associated with MHC class I molecules and derived from infection with a pathogen or from mutated antigens. In order to quantify in vivo this antigen-specific CD8⁺ T cell killing activity, we use the in vivo killing assay (IVKA). Here, we describe the protocol for the lysis of cells expressing a CD8⁺ T cell melanoma epitope of the hgp100_25-33 protein (KVPRNQDWL). C57BL/6 recipient mice, receive first target cells, prepared from naive congenic (CD45.1) C57BL/6 spleen cells pulsed with the hgp100_25-33 peptide and labeled with CFSE and of non-pulsed control cells labeled with Brilliant violet. One day later, the spleen cells of recipient mice are isolated and analyzed by FACS to measure the amount of CFSE cells and Brillant Violet (BV) cells. The percentage of lysis is calculated by the difference between CFSE versus BV.

Measuring the ability of antigen-specific CD8⁺ T cells to lyse their antigen in vivo is very important to evaluate the adaptive cytotoxic response induced against a pathogen or a tumor antigen.

In a narrow definition, virucidal activity represents the activity by which to interact with and physically disrupt viral particles. In a broad definition, it includes the activity by which to functionally inhibit (neutralize) viral infectivity without apparent morphological alterations of the viral particles. The viral infectivity can be measured in cell culture system by means of plaque assay, infectious focus assay, 50% tissue culture infectious dose (TCID₅₀) assay, etc. Morphologically, disruption of viral particles can be demonstrated by negative staining electron microscopic analysis of viral particles. In this article, we describe methods to assess virucidal activity in a broad definition.

Chemical and sedimentation procedures are used to purify virus particles. While these approaches are successful for wild-type viruses, they are often not feasible for purifying mutant viruses with assembly defects. We combined two published methods (Atasheva et al., 2013; Moller-Tank et al., 2013), to generate a protocol that uses low-speed centrifugation to purify both wildtype and mutant enveloped virus particles at high yield with minimal handling steps. This protocol has successfully been used to purify alphavirus particles for imaging and structural studies (Wang et al., 2015; Ramsey et al., 2017).

Due to its particulate material, mono-sodium urate (MSU) crystals are potent activators of the NOD-like receptor NLRP3. Upon activation, NLRP3 induces the formation of inflammasome complexes, which lead to the production and release of mature IL-1β. Bioactive IL-1β is a potent activator of innate immune responses and promotes recruitment of inflammatory cells, including neutrophils from the blood into damaged/inflamed tissues. This protocol describes a method to study in vivo inflammasome activation via intraperitoneal injection of MSU crystals. MSU-injection results in a drastic increase of intraperitoneal IL-1β levels, promoting neutrophil infiltration. Early-stage neutrophil numbers correlate with the amount of released IL-1β and can be used as a read-out for the extent of in vivo inflammasome activation. In addition, this protocol might also be used as a sterile peritonitis model, to investigate mechanisms of neutrophil recruitment to the peritoneum, or as a means to obtain large numbers of in vivo activated neutrophils.

This protocol describes a method for registration of in vivo cortical retinotopic map with cytochrome c oxidase (CO) labeled architectonic maps of the same mouse brain through the alignment of vascular fiducials. By recording surface blood vessel pattern and sequential alignment at each step, this method overcomes the challenge imposed by tissue distortion during perfusion, mounting, sectioning and histology procedures. This method can also be generalized to register and align other types of in vivo functional maps like ocular dominance map and spatial/temporal frequency tuning map with various anatomical maps of mouse cortex.

Notch signaling is an evolutionarily conserved signaling pathway that plays an indispensable role during development, and in the maintenance of homeostatic processes, in a wide variety of tissues (Kopan, 2012; Hori et al., 2013). The multifaceted roles of Notch signaling are stringently regulated at different levels. One of the most important aspects of regulation is the binding of different Notch ligands to each Notch receptor (NOTCH1-NOTCH4). Canonical ligands Delta or Serrate (in Drosophila), and Delta-like (DLL1 and DLL4) or Jagged (JAG1 and JAG2) (in mammals), are transmembrane glycoproteins. Ligands expressed on one cell bind to Notch receptors on an adjacent cell to induce Notch signaling. Glycosylation of Notch receptor extracellular domain by O-fucose and O-GlcNAc glycans is well established as critical regulators for Notch signaling strength (Stanley and Okajima, 2010; Haltom and Jafar-Nejad, 2015; Sawaguchi et al., 2017). In order to characterize Notch ligand binding to Notch receptors in isolated cells, we utilize Notch ligand extracellular domains tagged at the C-terminus by a human Fc domain, and determine binding of fluorescent anti-Fc antibody by flow cytometry.

Glioblastoma (GBM) is the most common primary brain cancer in adults and has a poor prognosis. It is characterized by a high degree of cellular infiltration that leads to tumor recurrence, atypical hyperplasia, necrosis, and angiogenesis. Despite aggressive treatment modalities, current therapies are ineffective for GBM. Mouse GBM models not only provide a better understanding in the mechanisms of gliomagenesis, but also facilitate the drug discovery for treating this deadly cancer. A retroviral vector system that expresses PDGFBB (Platelet-derived growth factor BB) and inactivates PTEN (Phosphatase and tensin homolog) and P53 tumor suppressors provides a rapid and efficient induction of glioma in mice with full penetrance. In this protocol, we describe a simple and practical method for inducing GBM formation by retrovirus injection in the murine brain. This system gives a spatial and temporal control over the induction of glioma and allows the assessment of therapeutic effects with a bioluminescent reporter.