The time-of-flight inflammasome evaluation (TOFIE) flow cytometric technique allows for the quantification of cells that contain specks. TOFIE, despite its advantages, is unable to perform single-cell analysis that includes the simultaneous observation of ASC speck locations, caspase-1 activity, and their detailed physical characteristics. This imaging flow cytometry procedure is described, providing a solution to these limitations. Inflammasome and Caspase-1 Activity Characterization and Evaluation (ICCE) employs the Amnis ImageStream X for rapid, single-cell, high-throughput image analysis, achieving an accuracy exceeding 99.5%. Quantitative and qualitative characterizations of ASC speck and caspase-1 activity's frequency, area, and cellular distribution are performed on mouse and human cells by ICCE.
Contrary to the prevalent notion of a static Golgi apparatus, it is, in reality, a dynamic entity, and a sensitive indicator of the cell's condition. Upon exposure to a variety of stimuli, the intact Golgi structure breaks down into smaller fragments. The resultant fragmentation can be either partial, creating multiple separated portions, or complete, leading to the complete vesiculation of the organelle. Varied morphological structures provide the basis for different techniques used to measure the Golgi complex's functional state. This chapter elucidates our flow cytometry-based imaging approach for determining changes in Golgi organization. Rapid, high-throughput, and robust, this method captures the key benefits of imaging flow cytometry, along with the ease of implementation and analysis it provides.
Imaging flow cytometry possesses the ability to span the existing divide between diagnostic procedures identifying key phenotypic and genetic alterations in the clinical evaluation of leukemia and other hematological malignancies or blood-borne disorders. Leveraging the quantitative and multi-parametric power of imaging flow cytometry, our Immuno-flowFISH approach has advanced the field of single-cell analysis. The immuno-flowFISH procedure has undergone full optimization to pinpoint chromosomal abnormalities like trisomy 12 and del(17p) that are clinically important, specifically within clonal CD19/CD5+ CD3- Chronic Lymphocytic Leukemia (CLL) cells, all within a single diagnostic test. The integrated methodology surpasses standard fluorescence in situ hybridization (FISH) in terms of both accuracy and precision. For CLL analysis, we offer a detailed immuno-flowFISH application, featuring a carefully documented workflow, technical instructions, and rigorous quality control criteria. A next-generation imaging flow cytometry approach may offer exceptional advancements and possibilities for a more thorough understanding of disease at the cellular level, benefiting both research and clinical laboratory applications.
Persistent particles found in consumer products, polluted air, and work environments are frequently encountered by humans, presenting a modern-day hazard and prompting ongoing research efforts. The persistence of particles in biological systems, often dictated by particle density and crystallinity, is strongly correlated with light absorption and reflection. These attributes facilitate the identification of numerous persistent particle types through laser light-based methods, including microscopy, flow cytometry, and imaging flow cytometry, dispensing with the need for extra labels. Post-in vivo study and real-world exposure analyses, this identification method facilitates the direct examination of persistent environmental particles within biological samples. Autoimmune retinopathy Fully quantitative imaging techniques and computing advancements have enabled the advancement of microscopy and imaging flow cytometry, allowing a plausible exploration of the detailed interactions and effects of micron and nano-sized particles on primary cells and tissues. A compilation of studies that exploit the marked light absorption and reflection attributes of particles to detect them within biological specimens is provided in this chapter. This section describes the methods used for the analysis of whole blood samples, encompassing imaging flow cytometry techniques for recognizing particles related to primary peripheral blood phagocytic cells, employing both brightfield and darkfield imaging.
The -H2AX assay is a sensitive and reliable procedure for determining the occurrence of radiation-induced DNA double-strand breaks. Manually analyzing individual nuclear foci using the conventional H2AX assay is a laborious and time-consuming process, making it inappropriate for high-throughput screening, especially when dealing with large-scale radiation accidents. Employing imaging flow cytometry, we have crafted a high-throughput H2AX assay. The method begins with sample preparation from small blood volumes within the Matrix 96-tube format. Automated image acquisition of -H2AX labeled cells, stained using immunofluorescence, is undertaken by ImageStreamX. This is then followed by the quantification and batch processing of -H2AX levels using the IDEAS software. A small blood sample enables the rapid analysis of -H2AX levels in several thousand cells to provide accurate and dependable quantitative measurements of -H2AX foci and mean fluorescence levels. This high-throughput -H2AX assay presents a valuable instrument, applicable not only to radiation biodosimetry during mass casualty incidents, but also to extensive molecular epidemiological investigations and personalized radiotherapy.
To determine the ionizing radiation dose received by an individual, biodosimetry methods measure exposure biomarkers within tissue samples from that person. Various expressions of these markers encompass DNA damage and repair mechanisms. A mass casualty incident involving radiological or nuclear material requires the immediate transmission of this information to medical responders, crucial for managing the potential exposure of affected victims. Microscopic analysis underpins traditional biodosimetry, leading to extended durations and substantial manual effort. To increase the processing speed of samples in the aftermath of a large-scale radiological mass casualty, several biodosimetry assays have been modified to suit the analysis capabilities of imaging flow cytometry. With a focus on the latest methodologies, this chapter provides a brief overview of these methods used to pinpoint and quantify micronuclei in binucleated cells of a cytokinesis-block micronucleus assay, utilizing an imaging flow cytometer.
Across a spectrum of cancer types, multi-nuclearity is a recurring attribute of the cellular structure. For a comprehensive assessment of drug toxicity, the observation of multinucleated cells in cultured cells is a frequently used analytical tool. Drug treatments and cancer frequently induce multi-nuclear cells due to flaws in cell division and cytokinesis. These cells, characteristic of advancing cancer, are often numerous and multi-nucleated, frequently correlating with a poor outcome. Automated slide-scanning microscopy results in improved data collection by minimizing bias in scoring processes. Despite its merits, this strategy suffers from limitations, such as the inability to effectively discern multiple nuclei within cells attached to the substrate at low magnification levels. The protocol for preparing multi-nucleated cell samples from attached cultures and the subsequent IFC analysis method are described in detail here. Multi-nucleated cells, products of both taxol-induced mitotic arrest and cytochalasin D-mediated cytokinesis blockade, can be imaged with maximal resolution through the IFC method. Two algorithms are devised for the purpose of discriminating between single-nucleus and multi-nucleated cells. Prosthetic knee infection We discuss the relative merits and demerits of immunofluorescence cytometry (IFC) and microscopy when applied to the examination of multi-nuclear cells.
Protozoan and mammalian phagocytes host the replication of Legionella pneumophila, the causative agent of Legionnaires' disease, a severe pneumonia, within a specialized intracellular compartment, the Legionella-containing vacuole (LCV). This compartment, in contrast to fusion with bactericidal lysosomes, exhibits substantial interaction with numerous cellular vesicle trafficking pathways, ultimately and tightly associating with the endoplasmic reticulum. A detailed comprehension of LCV formation hinges on the identification and kinetic analysis of cellular trafficking pathway markers within the pathogen vacuole. Imaging flow cytometry (IFC) is used in this chapter to provide a detailed description of the objective, high-throughput, and quantitative analysis of various fluorescently tagged proteins or probes on the LCV. To analyze Legionella pneumophila infection, we utilize Dictyostelium discoideum, a haploid amoeba, with the approach of examining fixed and complete infected host cells, or alternatively, LCVs from homogenized amoebae specimens. The contribution of a particular host factor to LCV formation is evaluated by comparing parental strains with their corresponding isogenic mutant amoebae. To quantify two LCV markers within intact amoebae or, alternatively, to identify LCVs with one probe while the other probe quantifies LCVs within host cell homogenates, amoebae concurrently generate two uniquely fluorescently tagged probes. Sulfopin price The IFC approach allows for the rapid generation of statistically robust data originating from thousands of pathogen vacuoles, and its application is feasible in other infection models.
The erythropoietic unit, known as the erythroblastic island (EBI), is a multicellular structure where a central macrophage fosters a circle of developing erythroblasts. For over half a century since the identification of EBIs, traditional microscopy methods, following sedimentation enrichment, remain the primary means of studying them. Quantification of EBI values and their frequency in the bone marrow or spleen is not enabled by the non-quantitative nature of these isolation procedures. Macrophage and erythroblast marker co-expression in cell aggregates has been quantified through flow cytometric means; however, determining if these aggregates also contain EBIs is not feasible, given the inability to visually assess their EBI content.