Protocol for immunophenotyping out-of-hospital cardiac arrest patients.

Immunophenotyping of out-of-hospital cardiac arrest (OHCA) patients is of increasing interest but has challenges. Here, we describe steps for the design of the clinical cohort, planning patient enrollment and sample collection, and ethical review of the study protocol. We detail procedures for blood sample collection and cryopreservation of peripheral blood mononuclear cells (PBMCs). We detail steps to modulate immune checkpoints in OHCA PBMC ex vivo. This protocol also has relevance for immunophenotyping other types of critical illness. For complete details on the use and execution of this protocol, please refer to Tamura et al. (2023).1.

Assessing mRNA translation in mouse adult microglia and bone-marrow-derived macrophages.

Protein synthesis, or mRNA translation, is the biological process through which genetic information stored in messenger RNAs is encoded into proteins. Here, we present an optimized protocol for assessing the translation rate in mouse adult microglia and cultured bone-marrow-derived macrophages. We describe steps for isolating cells, treating them with a puromycin-analog probe, and fluorescently labeling the puromycylated-polypeptide chains. We then detail their quantification by flow cytometry or with a fluorescent plate reader. For complete details on the use and execution of this protocol, please refer to Keane et al. (2021).1.

Characterizing CD4 T cell differentiation in mouse small intestine using T cell transfer, lamina propria preparation, and flow cytometry.

Studying gene function in T cells is crucial for understanding physiology and disease pathogenesis. Here, we provide a protocol to examine the role of specific genes in CD4+ T cell differentiation in the intestine. We describe steps for isolating naïve CD4+ T cells from mouse spleens and transferring them to recipient mice. We detail procedures to isolate lamina propria cells and analyze CD4+ T subsets using flow cytometry. This protocol is useful in the study of mucosal immune functions. For complete details on the use and execution of this protocol, please refer to Duan et al.1.

A protocol to detect human CD4+ T cell extracellular traps using scanning electron microscopy.

We present a protocol to detect extracellular traps (ETs) induced by Cutibacterium acnes in cultured TH17 clones. We first describe the isolation of C. acnes-specific TH17 clones by sterile cell sorting. We then detail the in vitro induction of ETs in TH17 clones stimulated by C. acnes and the imaging of released ETs using scanning electron microscopy. This protocol can be applied to the study of other ETs released by other T cell subsets. For complete details on the use and execution of this protocol, please refer to Agak et al. (2021).1.

CyTOF protocol for immune monitoring of solid tumors from mouse models.

Techniques for robust immune profiling of mouse tumor and blood are key to understanding immunological responses in mouse models of cancer. Here, we describe mass cytometry (cytometry by time-of-flight) procedures to facilitate high-parameter profiling of low-volume survival blood samples and end-of-study tumor samples. We employ live-cell barcoding systems to mark all cells from each tumor and blood to improve cost-effectiveness and minimize batch effects. For complete details on the use and execution of this protocol, please refer to Charmsaz et al. (2021).1.

Dissociation and flow cytometric isolation of murine intestinal epithelial cells for multi-omic profiling.

Intestinal epithelium is composed of several cell types, which can be dissociated but difficult to maintain high cell viability due to anoikis. Herein, we describe a step-by-step protocol for the isolation of highly viable intestinal epithelial cells using ethylenediaminetetraacetate acid and TrypLE Express, which can subsequently be employed for multi-omic analyses, including single-cell RNA sequencing. For complete details on the use and execution of this protocol, please refer to Ge et al. (2022).1.

Viral competition assay to assess the role of HIV-1 proteins in immune evasion.

CD8+ T lymphocytes can recognize and eliminate cells infected by viruses. However, the human immunodeficiency virus (HIV-1) has developed mechanisms to evade CD8+ T-cell-mediated clearance. Here, we describe a protocol to assess the role of the HIV-1 protein Nef in immune evasion. The viral competition assay reveals the preferential killing of HIV-1-infected cells unable to express Nef. This methodology can be extended to study HIV-1 proteins involved in immune evasion and viral variants encoding cytotoxic T lymphocyte escape mutations. For complete details on the use and execution of this protocol, please refer to Duette et al. (2022).1.

Flow cytometry protocol to quantify immune cells in the separated epithelium and stroma of herpes simplex virus-1-infected mouse cornea.

Existing flow cytometry approaches identify immune cells using the whole infected/inflamed cornea, which limits its ability to distinguish the immune cells infiltrating the corneal epithelium from the corneal stroma. Here, we present a protocol to analyze immune cells in the separated epithelium and stroma from naïve and herpes simplex virus-1 (HSV-1)-infected mouse corneas. We describe steps for viral infection, separation of corneal epithelium from stroma, preparation of a single-cell suspension of the individual epithelium and stroma, and flow cytometry assay.

Longitudinal tracking of T cell lymphomas in mice using flow cytometry.

T cell hematological cancer has a complex interplay with host immune cells, but the ability to experimentally discriminate transferred cancer cells from host cells by flow cytometry is technically challenging. Here, we present a flow cytometry protocol to evaluate cancer cell and host immune phenotypes following transplant of a T cell lymphoma bearing a congenic marker (CD45.2) into a syngeneic host (CD45.1). We describe steps for isolation of primary immune cells from mice, staining preparation with flow cytometry antibody cocktails, and analysis by flow cytometry. For complete details on the use and execution of this protocol, please refer to Kuczynski et al.1.

Protocol for identification and computational analysis of human natural killer cells using flow cytometry and R.

Identifying differential protein expression is routinely used to delineate natural killer (NK) cells from various sample cohorts. This protocol describes key steps for NK cell analysis: identifying human NK cells using flow gating, data export from FlowJo, data loading in R, dimensionality reduction and visualization with Uniform Manifold Approximation and Projection, and generalized linear modeling with CyotGLMM. These analyses can help generate potential biomarkers of interest to identify NK cells across aging, treatment groups, and others. For complete details on the use and execution of this protocol, please refer to Kroll et al. (2022).1.

Chimerism and phenotypic analysis of intraepithelial and lamina propria T cells isolated from human ileal biopsies after intestinal transplantation.

Understanding immune cell dynamics after intestinal transplantation has provided new insights into human lymphocyte biology. However, isolating and characterizing such cells can be challenging. Here, we provide a protocol to isolate intraepithelial and lamina propria lymphocytes from human ileal biopsies. We describe techniques for flow cytometric analysis and determination of multilineage chimerism and T lymphocyte phenotypes. This protocol can be modified to isolate and analyze lymphocytes from other tissues. For complete details on the use and execution of this protocol, please refer to Fu et al. (2019)1 and Fu et al. (2021).2.

Engineering the sialome of mammalian cells with sialic acid mimetics.

Mammalian glycans show a diversity in sialic acid capping, constituting the sialome. Sialic acids can be extensively modified chemically, yielding sialic acid mimetics (SAMs). Here, we present a protocol for detecting and quantifying incorporative SAMs using microscopy and flow cytometry, respectively. We detail steps for linking SAMS to proteins with western blotting. Lastly, we detail procedures for incorporative or inhibitory SAMs and how SAMs can be used for the on-cell synthesis of high-affinity Siglec ligands. For complete details on the use and execution of this protocol, please refer to Büll et al.1 and Moons et al.2.

Protocol to analyze chromatin-bound proteins through the cell cycle using Chromoflow flow cytometry.

Chromatin-bound proteins have been conventionally measured through subcellular fractionation followed by immunoblotting or by immunofluorescence microscopy. Here, we present Chromoflow, a protocol for the quantitative analyses of protein levels on chromatin in single cells and throughout the cell cycle using flow cytometry. We describe steps for harvesting cells and for nuclear extraction, and a barcoding strategy to multiplex samples from different conditions that reduces antibody staining variability and eliminates the need for normalization.1,2 We then detail procedures for data acquisition and analysis. For complete details on the use and execution of this protocol, please refer to Alonso-Gil et al. (2023).3.

FluoroSpot assay to analyze SARS-CoV-2-specific T cell responses.

Monitoring antigen-specific T cell frequency and function is essential to assess the host immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we present a FluoroSpot assay for concurrently detecting ex vivo antiviral cytokine production by SARS-CoV-2-specific T cells following peptide stimulation. We then detail intracellular cytokine staining by flow cytometry to further validate the FluoroSpot assay results and define the specific T cell subpopulations. For complete details on the use and execution of this protocol, please refer to Tiezzi et al. (2023).1.

Generation, culture, and stimulation of small intestinal murine organoids in parasitology research.

Parasitic helminth worms frequently infect the gastrointestinal tract and interact with the intestinal epithelium and specialized cell types within it. Intestinal organoids derived from stem cells that line the intestine represent a transformational technology in the study of epithelial-parasite dialogue. Here, we present a protocol for establishing small intestine organoid cultures and administering parasite products of interest to these cultures. We then describe steps for evaluating their impact by microscopy, flow cytometry, immunohistology, and mRNA gene expression. For complete details on the use and execution of this protocol, please refer to Drurey et al. (2022).1.

Protocol for neonatal respiratory syncytial virus infection in mice and immune analysis of infected lungs and bronchoalveolar lavage fluid.

Respiratory syncytial virus (RSV) infection in infants and toddlers is a major public health problem. Here, we provide a protocol for neonatal RSV infection in mice and immune analysis of infected lungs and bronchoalveolar lavage (BAL) fluid. We describe steps for anesthesia and intranasal inoculation, weight monitoring, and whole lung collection. We then detail BAL fluid immune and whole lung analyses. This protocol can be used for neonatal pulmonary infection with other viruses or bacteria.

Quantification of membrane-bound cytokine receptors by calibrated flow cytometry.

We present a protocol for quantifying the expression of the receptor gp130 using a calibrated flow cytometric approach. We describe pitfalls for receptor quantification such as titration of primary antibodies and standardizing cell culture. Receptors are stained with primary antibodies and fluorophore-coupled secondary antibodies. Beads covered with defined numbers of immunoglobulin G stained with fluorophore-coupled secondary antibodies serve as calibrators. In this way, the fluorescence intensity of cells is converted to the number of receptors on the cell surface. For complete details on the use and execution of this protocol, please refer to Reeh et al. (2019).1.

Flow cytometric analysis of phosphatidylcholine metabolism using organelle-selective click labeling.

Here, we present a protocol to analyze phosphatidylcholine (PC) metabolism in mammalian cells using organelle-selective click labeling coupled with flow cytometry (O-ClickFC). We describe steps for the metabolic incorporation of azide-choline into PC. We then detail fluorescent labeling of the azide-modified PC with organelle-targeting clickable dyes in the ER-Golgi, plasma membrane, and mitochondria, and by flow cytometry. This protocol is optimized for flow cytometric quantification of the labeled PC at the organelle level within single live cells. For complete details on the use and execution of this protocol, please refer to Tsuchiya et al. (2023).1.

Protocol for density gradient neutrophil isolation and flow cytometry-based characterization from human peripheral blood.

Neutrophils are the first immune responders to bacterial or viral infection and play key roles in the host immune response; however, handling and investigating fresh neutrophils can be challenging. Here, we present a protocol for isolating neutrophils from the peripheral blood of healthy donors using density gradient separation method. We describe steps for morphology analysis by cytospin and immunophenotyping by flow cytometry analysis. This protocol can be used for the isolation of neutrophils from healthy and diseased individuals. For complete details on the use and execution of this protocol, please refer to Parthasarathy et al.1.

Retro-orbital CD45 antibody labeling to evaluate antibody-secreting cell trafficking in mice.

Antibody-secreting cells (ASCs) are critical regulators of the humoral immune response. However, differences between tissue resident populations versus those that have recently migrated to their final anatomic destination are poorly understood. Here, we present a protocol for using retro-orbital (r.o.) CD45 antibody labeling to identify tissue resident versus recently immigrated ASCs in mice. We describe steps for r.o. injection of antibodies, animal euthanasia, and tissue harvesting. We then detail tissue processing, cell counting, and cell staining for flow cytometry analysis. For complete details on the use and execution of this protocol, please refer to Pioli et al. (2023).1.