Methods in cell biology最新文献

Broadly speaking, cell tracking dyes are fluorescent compounds that bind stably to components on or within the cells so the fate of the labeled cells can be followed. Their staining should be bright and homogeneous without affecting cell function. For purposes of monitoring cell proliferation, each time a cell divides the intensity of cell tracking dye should diminish equally between daughter cells. These dyes can be grouped into two different classes. Protein reactive dyes label cells by reacting covalently but non-selectively with intracellular proteins. Carboxyfluorescein diacetate succinimidyl ester (CFSE) is the prototypic general protein label. Membrane intercalating dyes label cells by partitioning non-selectively and non-covalently within the plasma membrane. The PKH membrane dyes are examples of lipophilic compounds whose chemistry allows for their retention within biological membranes without affecting cellular growth, viability, or proliferation when used properly. Here we provide considerations based for labeling cell lines and peripheral blood mononuclear cells using both classes of dyes. Examples from optimization experiments are presented along with critical aspects of the staining procedures to help mitigate common risks. Of note, we present data where a logarithmically growing cell line is labeled with both a protein dye and a membrane tracking dye to compare dye loss rates over 6days. We found that dual stained cells paralleled dye loss of the corresponding single stained cells. The decrease in fluorescence intensity by protein reactive dyes, however, was more rapid than that with the membrane reactive dyes, indicating the presence of additional division-independent dye loss.

Next to conventional cancer therapies, immunotherapies such as immune checkpoint inhibitors have broadened the cancer treatment landscape over the past decades. Recent advances in next generation sequencing and bioinformatics technologies have made it possible to identify a patient's own immunogenic neoantigens. These cancer neoantigens serve as important targets for personalized immunotherapy which has the benefit of being more active and effective in targeting cancer cells. This paper is a step-by-step guide discussing the different analyses and challenges encountered during in-silico neoantigen prediction. The protocol describes all the tools and steps required for the identification of immunogenic neoantigens.

Lysosomal acid lipase (LAL) is a key enzyme in the metabolic pathway of neutral lipids, whose deficiency (LAL-D) induces the differentiation of myeloid lineage cells into myeloid-derived suppressor cells (MDSCs), which promotes tumor growth and metastasis. This protocol provides detailed procedures for assessment of various LAL biochemical and physiological activities in Ly6G⁺ and CD11c⁺ MDSCs, including isolation of Ly6G⁺ and CD11c⁺ cells from the bone marrow and blood of mice, assays of LAL-D-induced cellular metabolic and mitochondrial activities, assessment of LAL-D-induced pathogenic immunosuppressive activity and tumor stimulatory activity. Pharmacological inhibition of the LAL activity was also described in both murine myeloid cells and human white blood cells.

Myeloid-derived suppressor cells (MDSCs) comprise a heterogeneous population of myeloid origin and immature state, whose hallmark is the capacity to suppress T cells and other immune populations. In mice, the first approach to identify MDSCs relies in the measurement of their phenotypical markers: CD11b and GR-1. In addition, two main subtypes of MDSCs have been defined based on the expression of the following markers: CD11b+ Ly6G- Ly6C+ (monocytic-MDSCs, M-MDSCs) and CD11b+ Ly6G+ Ly6C^+/low (polymorphonuclear-MDSCs, PMN-MDSCs). Since CD11b+ GR-1+ (Ly6C+/Ly6G+) MDSCs can increase significantly in peripheral blood during numerous acute or chronic processes, measuring alterations in the phenotypic markers CD11b and GR-1 could be important as a first step before assessing the suppressive function of the cells. In many cases it could be necessary to measure CD11b+ Gr-1+ cells from a minimum volume of peripheral blood cells without greatly affecting animal viability, since this approach would allow for further studies to be conducted on subsequent days, such as measuring parameters of the immune response or even survival in the context of the pathology under study. The following protocol describes a simple and optimized protocol for measuring the presence of CD11b+ GR-1+ (Ly6C+/Ly6G+) myeloid cells using 2+ channel flow cytometry, from a minimum volume of mouse peripheral blood obtained by facial vein puncture.

Numerous studies have shown that aging in humans leads to a decline in olfactory function, resulting in deficits in acuity, detection threshold, discrimination, and olfactory-associated memories. Furthermore, impaired olfaction has been identified as a potential indicator for the onset of age-related neurodegenerative diseases, including Alzheimer's disease (AD). Studies conducted on mouse models of AD have largely mirrored the findings in humans, thus providing a valuable system to investigate the cellular and circuit adaptations of the olfactory system during natural and pathological aging. However, the majority of previous research has focused on assessing the detection of neutral or synthetic odors, with little attention given to the impact of aging and neurodegeneration on the recognition of social cues-a critical feature for the survival of mammalian species. Therefore, in this study, we present a battery of olfactory tests that use conspecific urine samples to examine the changes in social odor recognition in a mouse model of neurodegeneration.

Traumatic brain injury (TBI) represents one of the leading causes of disability and death worldwide. The annual economic impact of TBI-including direct and indirect costs-is high, particularly impacting low- and middle-income countries. Despite extensive research, a comprehensive understanding of the primary and secondary TBI pathophysiology, followed by the development of promising therapeutic approaches, remains limited. These fundamental caveats in knowledge have motivated the development of various experimental models to explore the molecular mechanisms underpinning the pathogenesis of TBI. In this context, the Lateral Fluid Percussion Injury (LFPI) model produces a brain injury that mimics most of the neurological and systemic aspects observed in human TBI. Moreover, its high reproducibility makes the LFPI model one of the most widely used rodent-based TBI models. In this chapter, we provide a detailed surgical protocol of the LFPI model used to induce TBI in adult Wistar rats. We further highlight the neuroscore test as a valuable tool for the evaluation of TBI-induced sensorimotor consequences and their severity in rats. Lastly, we briefly summarize the current knowledge on the pathological aspects and functional outcomes observed in the LFPI-induced TBI model in rodents.

Cells are dynamic machines that continuously change their architecture to adapt and respond to extracellular and intracellular stimuli. Deciphering dynamic processes with nanometer-scale resolution inside cells is critical for mechanistic understanding. Here, we present a protocol that enables the in situ study of dynamic changes in intracellular structures under close-to-native conditions at high spatiotemporal resolution. Importantly, the cells are grown, transported, and imaged in a chamber in which environmental conditions such as temperature and gas (e.g., carbon dioxide or oxygen) concentration can be controlled. We demonstrate this protocol to quantify ultrastructural changes that occur during the cell cycle of cultured mammalian cells. The environment control system opens up the possibility of applying this method to primary cells, tissues, and organoids by adjusting environmental conditions.

Brain metastases (BrM) occur when malignant cells spread from a primary tumor located in other parts of the body to the brain. BrM is a deadly complication for cancer patients and severely lacks effective therapies. Due to the limited access to patient samples, preclinical models remain a very valuable tool for studying metastasis development, progression, and response to therapy. Thus, reliable methods to assess metastatic burden in these models are crucial. Here we describe step by step a new semi-automatic machine-learning approach to quantify metastatic burden on mouse whole-brain stereomicroscope images while preserving tissue integrity. This protocol uses the open-source and user-friendly image analysis software QuPath. The method is fast, reproducible, unbiased, and gives access to data points not always accessible with other existing strategies.

Despite being tightly regulated, messenger RNA (mRNA) translation, a manner in which cells control expression of genes and rapidly respond to stimuli, is highly dysfunctional and plastic in pathologies including cancer. Conversely, the investigation of molecular mechanisms whereby mRNA translation becomes aberrant in cancer, as well as inhibition thereof, become critical in developing novel therapeutic approaches. More specifically, in malignancies such as chronic lymphocytic leukemia in which aberrant global and transcript specific translation has been linked with poorer patient outcomes, targeting translation is a relevant approach, with various translation inhibitors under development. Here we elaborate on a protein synthesis assay by flow cytometry, O-propargyl-puromycin, demonstrating global mRNA translation rate with a variety of different applications including cell lines, primary cells or co-culture systems in vitro. This method provides a comprehensive tool in quantifying the rate of global mRNA translation in cancer cells, as well as that of the tumor microenvironment cells, or in response to inhibitory therapeutic agents while offering the possibility to simultaneously assess other cellular markers.

To develop new effective therapeutic strategies for cancer patients, there is a need for extensive and precise insights into the mechanisms involved in the immune response to anti-cancer treatments. The enzyme-linked immunospot (ELISpot) assay is a rapid and reproducible technique that allows the detection of cytokine-producing antigen-specific T cells at the single cell level. This protocol describes an interferon gamma (IFN-γ) ELISpot method for measuring antigen-specific murine CD8⁺ T cells that produce IFN-γ, a marker of their activation and cytotoxicity. Splenocytes from tumor-bearing mice treated with radiation therapy were used as source of CD8⁺ T cells and were stimulated with a tumor-derived peptide. This method was facilitated by a ready-to-use assay kit and provides a tool to analyze the specificity, intensity, and kinetics of specific CD8⁺ T cells.