Methods in cell biology最新文献

Phospho-flow is an invaluable tool for investigation into immunological signaling, enabling concurrent staining of cell lineage markers alongside sensitive intracellular phospho-proteins. Phospho-flow holds promise as a powerful diagnostic and therapeutic tool that can enable signal profiling, cell phenotyping, drug screening, pharmacodynamic profiling and assessment of drug efficacy across multiple cell types. When combining phospho-flow with fluorescent cell barcoding (FCB) multiplexing technique, high throughput flow cytometry can be achieved. FCB enhances experiment robustness while reducing variability in staining and decreasing antibody usage. Despite its utility, inter-operator technique variability persists, highlighting the need for protocol and analysis standardization. Here we describe an experimental mechanism for stimulating mouse and human T cells that demonstrates robust activation that can be adapted for various experimental designs.

T-cell redirecting therapies such as chimeric antigen receptor (CAR) T cells and bispecific antibodies (bsAbs) creating an immunological synapse between target and effector cells are at the forefront of cancer immunotherapies. Selective pressure by these therapies can cause the enrichment of antigen negative (Ag)^low/^- tumor cell variants which are resistant to therapy and may drive clinical relapse, i.e. Ag escape. Ag^low/- cells generally exist in the tumor tissue prior to immunotherapies. Therefore, Ag escape is an integral part of tumor survival and poses a significant hurdle to overcome. Here we describe an approach used to quantify and functionally assess Ag^- tumor cells. This method enables high-throughput screening of single-cell biopsy or PBMC samples and allows for the assessment of their propensity to be targeted in a straightforward in vitro assay. Methods described here can be modulated and be incorporated to study samples derived from different anatomical locations and can be incorporated in a larger panel of flow cytometry for extensive phenotyping.

The immune system plays a critical role in a number of pathologic conditions, including infection, autoimmunity, and cancer. The migratory capacity of immune cells is essential to their ability to reach and invade into sites of infection, tissue damage, or solid tumors. Notably, studying the migration and invasion of immune cells has become increasingly important in the treatment of cancer. Immunotherapy has revolutionized the treatment of cancer. One critical barrier to the efficacy of immunotherapy, especially in solid tumors, is the ability of immune cells, specifically T and Natural Killer (NK) cells, to infiltrate into the tumor microenvironment (TME). Because of this, it has become increasingly important to study the invasive and migratory capabilities of immune cells in the context of cancer. Additionally, in vitro models that better recapitulate the TME are necessary in order to examine the invasion of human immune cells. Here, we describe 2D and 3D methods that can be used to examine the migratory and invasive capabilities of immune cells. These techniques have been adapted from previously described techniques.

S-palmitoylation of cysteine residues is the only lipid-based posttranslational modification of proteins that is reversible and therefore has important implications in cellular function. S-palmitoylation has been associated with several cellular processes (e.g., cell signaling, protein transport, cell cycle, immune response, lipid metabolism, host-pathogen interaction) and human diseases, including neurological disorders, cancer, and infectious diseases. However, S-palmitoylation research has been hampered by the cumbersome experimental protocols necessary for its study. Currently, there are two main methodologies that, coupled with mass spectrometry (MS), allow the study of S-palmitoylated proteins proteome-wide. They mainly differ in the way of labeling palmitoylated proteins: one relies on "metabolic labeling" with a palmitic acid analog in living cells, while the other is based on "chemical labeling" of thiol groups derived from palmitoylated sites in extracted proteins. Although metabolic labeling is restricted to cultured cells, we will focus on this technique as it is more sensitive and specific than others. Here, we describe the protocol to measure palmitoylation in cancer cells using metabolic labeling coupled to SILAC-based mass spectrometry quantification, which can be applied to other mammalian cell models. Facilitating the use of this methodology will extend the knowledge of palmitoylation signaling and unravel potential therapeutic avenues for diseases in which this unexplored modification is implicated.

Myeloid-derived suppressor cells (MDSC) are heterogenous group of immature and mature myeloid cells that accumulate in chronic inflammatory conditions and contribute to the immune suppression. Their ability to inhibit immune responses makes them critical targets for therapeutic interventions. However, working with patient-derived MDSC poses challenges. Variability in cell yields and the complexity of their isolation procedures significantly hinder the conduct of in vitro experiments aimed at investigating potential targeting strategies. In this chapter, we introduce a method to generate human CD14⁺ MDSC-like cells in vitro from healthy donor monocytes. This method provides a consistent and reproducible framework for studying MDSC functions and testing potential inhibitors of their activity.

Liquid biopsy is a non-invasive molecular test that uses biofluid to detect cancer. This is a rapidly growing field that encompasses a diverse class of assays that rely on circulating nucleic acids, proteins, and cells to identify cancer. Recently, there is particular interest in using circulating tumor DNA (ctDNA) to screen for cancer, to monitor treatment response, and to help guide therapeutic decisions. Here, we will review the procedural aspects of ctDNA assays, as well as the various applications of ctDNA assays in cancer care. The utility of various biofluids to conduct ctDNA assays, the technical considerations for performing the assays, and several possible available assay formats that can be used for ctDNA analysis are reviewed. Several protocols that can be used for ctDNA isolation, detection, and quantification are presented, including targeted ctDNA qPCR.

T cell receptor (TCR) signaling strength influences critical T cell characteristics including cytotoxic capacity, differentiation, memory formation, and exhaustion. Naturally occurring or engineered single-nucleotide variants (SNVs) and gene deletions which modulate T cell signaling pathways can significantly impact the potency of T cell cytolytic activity, including in the setting of T cell-based immunotherapies such as tumor-infiltrating lymphocyte (TIL) therapy and chimeric antigen receptor T cell (CAR T) therapy. Thus, studying T cell signaling represents a valuable component of the engineering and preclinical testing process for cell therapies and immune checkpoint blockade (ICB). Flow cytometry is a powerful experimental and diagnostic tool which enables rapid, quantitative analysis of T cell signaling responses by phosphoprotein detection with fluorophore-labeled antibodies. However, many approaches that have been developed to induce and study T cell signaling rely on supraphysiological and non-specific stimulation methods, such as crosslinking of T cell CD3 and CD28 or treatment with chemical stimulating agents such as ionomycin and phorbol 12-myristate 13-acetate (PMA). These assays bypass the endogenous TCR machinery and limit the conclusions which can be drawn regarding the physiological relevance of the T cell responses measured. Here, we present a simple, efficient, and scalable workflow to assess physiological T cell signaling responses to antigen-specific stimulation with cognate peptide-MHC expressing target cells using flow cytometry. With minimal modifications, this approach can be successfully applied to the study of chimeric antigen receptor (CAR) signaling or signaling in other immune cell subtypes such as B cells, using analogous antigen-matched co-culture systems.

Detection of cytosolic double-stranded DNA (dsDNA) is critical for understanding its role in innate immunity, infection, cancer, and autoimmune diseases. Various methods have been developed to identify and quantify cytosolic dsDNA, each offering unique strengths in terms of sensitivity, specificity, and throughput. Immunofluorescence microscopy enables direct visualization of cytosolic dsDNA, providing insights into its localization and dynamics within cells. Enzyme-linked immunosorbent assays (ELISAs) and immunoblotting techniques detect dsDNA indirectly through associated proteins like cGAS. Fluorescent spectroscopy-based methods allow for rapid and specific quantification of dsDNA. Additionally, biosensors and nanotechnology-based approaches are emerging as novel tools for dsDNA detection with enhanced sensitivity. This methodological compendium provides an overview of the common lab bench methodologies, highlighting their applications, limitations, and potential advancements in the detection of cytosolic dsDNA in various sample forms, such as cells, sectioned tissue, and cellular cytosol extracts.

Here, we present a comprehensive and reproducible protocol for the isolation, cryopreservation, and immunophenotyping of human peripheral blood mononuclear cells (PBMCs) using a 28-color flow cytometry panel. This high-dimensional panel enables the simultaneous assessment of conventional and unconventional T cells and their activation or functional states in a single sample, making it a valuable tool for translational and clinical immunology research. This protocol outlines detailed procedures for blood processing, density gradient separation, and optimized washing steps to minimise platelet contamination. Standardized cryopreservation methods are provided to facilitate longitudinal studies or multicentre trials. The staining strategy is specifically optimized for PBMCs and is compatible with both conventional and spectral flow cytometry systems, allowing robust and reproducible identification of lymphocyte populations with a focus on T lymphocytes. This method is applicable to both fresh and cryopreserved PBMCs and has been validated on material from healthy donors and patients. It provides a reliable framework for immune monitoring in contexts such as infection, cancer, or immunotherapy. Additionally, this protocol includes key troubleshooting steps, guidance on panel design, and practical advice for data acquisition and analysis. The workflow is adaptable to other staining panels and can seamlessly be integrated into standardized immune profiling pipelines.

One important strategy of cells to communicate with their environment is the release of proteins which can serve as signals for other cells nearby or at distant places in a complex organism. A first step in the characterization and investigation of cell-cell communication in this context is to figure out which proteins are released from cells under defined experimental conditions. Here, we present an approach that will give rise to a high-quality secretome and detects proteins that will be confidently released by cultured cells. This approach is based on the separate preparation of proteins from conditioned medium and corresponding cell lysates. After protein digestion and quantitative mass spectrometric analysis, protein abundances are compared and proteins showing a significantly higher abundance in the secretome are identified. We assume that these proteins have a higher probability of being released by well-directed processes and not simply by contamination of (dead) cells. We show an optimized protocol in which samples from primary human normal dermal fibroblasts (NHDF) are prepared with the single-pot solid-phase-enhanced sample preparation (SP3) method from only 450μL conditioned medium along with one-hour gradient separations and data-independent mass spectrometric data acquisition.