Cells containing specks can also be enumerated by means of a flow cytometric technique, time-of-flight inflammasome evaluation (TOFIE). 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. We illustrate how an imaging flow cytometry technique circumvents these constraints. With over 99.5% accuracy, ICCE, a high-throughput, single-cell, rapid image analysis method using the Amnis ImageStream X instrument, characterizes and evaluates inflammasome and Caspase-1 activity. 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.
While the Golgi apparatus is often perceived as a stationary structure, it is actually a dynamic entity, and a delicate detector of the cell's state. Various stimuli trigger the fragmentation of the whole Golgi apparatus. This fragmentation may either partially fragment the organelle, resulting in several disconnected sections, or completely transform the organelle into vesicles. The differing morphologies of these structures form the groundwork for multiple techniques used to assess the Golgi apparatus's state. Our imaging flow cytometry methodology, detailed in this chapter, quantifies changes in Golgi structure. The method under consideration inherits imaging flow cytometry's strengths: speed, high-throughput capacity, and resilience. Furthermore, the method simplifies implementation and analytical procedures.
The ability of imaging flow cytometry is to close the gap presently existing between diagnostic tests that detect essential phenotypic and genetic changes in the clinical evaluation of leukemia and other hematological cancers or blood disorders. Our Immuno-flowFISH technique, using imaging flow cytometry's quantitative and multi-parametric power, has enabled us to extend the limitations of single-cell analysis. The optimization of the immuno-flowFISH technique allows for the detection of clinically consequential numerical and structural chromosomal abnormalities, including trisomy 12 and del(17p), within clonal CD19/CD5+ CD3- Chronic Lymphocytic Leukemia (CLL) cells in a single testing procedure. The integrated methodology displays greater accuracy and precision than the typical fluorescence in situ hybridization (FISH) technique. A detailed immuno-flowFISH application for CLL analysis, including a meticulously cataloged workflow, comprehensive technical instructions, and quality control considerations, is presented. This revolutionary imaging flow cytometry protocol promises groundbreaking progress and unique advantages for comprehensive cellular disease assessments, advantageous for both research and clinical labs.
Consumer products, air pollution, and work environments are sources of persistent particle exposure to humans, a current concern prompting active research. Light absorption and reflectance are significantly influenced by particle density and crystallinity, which in turn frequently determine the longevity of these particles within biological systems. Laser light-based techniques, including microscopy, flow cytometry, and imaging flow cytometry, enable the identification of several persistent particle types without requiring supplementary labels, thanks to these attributes. The direct analysis of environmental persistent particles in biological samples associated with in vivo studies and real-life exposures is enabled by this identification method. 6-Aminonicotinamide Thanks to the progress of fully quantitative imaging techniques and computing capabilities, microscopy and imaging flow cytometry have advanced, allowing a plausible account of the intricate interactions and effects of micron and nano-sized particles with primary cells and tissues. This chapter synthesizes research that uses particles' substantial light absorption and reflectance to locate them in biological specimens. The analysis of whole blood samples, accompanied by detailed imaging flow cytometry methods to identify particles alongside primary peripheral blood phagocytic cells, is presented using brightfield and darkfield parameters, is detailed next.
The radiation-induced DNA double-strand breaks can be assessed with high sensitivity and reliability using the -H2AX assay. Individual nuclear foci in the conventional H2AX assay are laboriously and painstakingly identified manually, which creates an inherently time-consuming process, thus obstructing its use in high-throughput screening, crucial for large-scale radiation accidents. Utilizing imaging flow cytometry, we have created a high-throughput system for H2AX detection and analysis. Sample preparation from reduced blood volumes, utilizing the Matrix 96-tube format, initiates this method. The procedure continues with the automated imaging of immunofluorescence-labeled -H2AX stained cells via ImageStreamX. Finally, the Image Data Exploration and Analysis Software (IDEAS) quantifies -H2AX levels and performs batch processing. The rapid analysis of -H2AX levels within several thousand cells, drawn from a small volume of blood, permits accurate and dependable quantitative measurements for -H2AX foci and average fluorescence intensity. A valuable tool, the high-throughput -H2AX assay's applications span radiation biodosimetry in mass casualty events, alongside vast-scale molecular epidemiological research and personalized radiotherapy.
Biomarkers of exposure, measured in tissue samples from an individual, are utilized by biodosimetry methods to determine the dose of ionizing radiation received. Markers, including processes of DNA damage and repair, find expression in diverse ways. To ensure appropriate medical care for victims potentially exposed to radiation or nuclear materials during a mass casualty event, the information must be rapidly communicated to medical personnel. Traditional biodosimetry methods, predicated on microscopic examination, suffer from the shortcomings of prolonged processing times and high labor requirements. In the wake of a large-scale radiological mass casualty event, multiple biodosimetry assays have been optimized for high-throughput analysis using imaging flow cytometry, enhancing sample turnaround time. A succinct review of these methods, emphasizing the most recent methodology for discerning and calculating micronuclei in binucleated cells of the cytokinesis-block micronucleus assay, is presented in this chapter using an imaging flow cytometer.
A prevalent trait in cancerous cells across diverse types of tumors is multi-nuclearity. Multi-nuclearity in cultured cells serves as a widely-used indicator of drug toxicity, facilitating assessments across various chemical compounds. In cancer and under the influence of drug treatments, multi-nuclear cells emerge from mistakes within the processes of cell division and cytokinesis. The presence of these cells, a hallmark of cancer progression, is often accompanied by an abundance of multinucleated cells, which frequently correlates with a poor prognosis. Automated slide-scanning microscopy offers a method to mitigate scorer bias and enhance the efficiency of data acquisition. While this procedure possesses strengths, it is constrained by factors like poor visualization of multiple nuclei in cells anchored to the substrate when using low magnification. The experimental procedure for isolating multi-nucleated cells from cultured samples, along with the IFC analysis protocol, is detailed below. Images of multi-nucleated cells, stemming from taxol-induced mitotic arrest and subsequent cytochalasin D-mediated cytokinesis blockade, are readily acquirable at the highest resolution of the IFC system. Two algorithms are suggested to classify cells as either single-nucleus or multi-nucleated. Medical geology We explore the benefits and drawbacks of immunocytochemistry-based analysis of multi-nucleated cells when compared to conventional microscopy techniques.
Replicating within protozoan and mammalian phagocytes, Legionella pneumophila, the causative agent of Legionnaires' disease, a severe pneumonia, does so inside a specialized intracellular compartment, the Legionella-containing vacuole (LCV). This compartment, instead of fusing with bactericidal lysosomes, engages in extensive interaction with various cellular vesicle trafficking pathways, ultimately and directly connecting to the endoplasmic reticulum. Understanding the complex mechanics of LCV formation depends critically on identifying and analyzing the kinetics of cellular trafficking pathway markers on the pathogen vacuole. The chapter explicates the use of imaging flow cytometry (IFC) for the objective, quantitative, and high-throughput measurement of different fluorescently tagged proteins or probes present on the LCV. In our study of Legionella pneumophila infection, we employ the haploid amoeba Dictyostelium discoideum, and investigate either fixed, complete infected host cells or LCVs from homogenized amoebae. To determine the influence of a particular host factor on LCV formation, a comparison is made between parental strains and isogenic mutant amoebae. Two different fluorescently tagged probes are simultaneously produced by the amoebae, enabling the tandem quantification of two LCV markers within intact amoebae, or the identification of LCVs using one probe and the quantification of the other probe in homogenized host cells. Global medicine Rapidly generating statistically robust data from thousands of pathogen vacuoles is possible with the IFC approach, and its application is viable for other infection models.
The erythroblastic island (EBI), a multicellular functional erythropoietic unit, consists of a central macrophage that nourishes a circle of developing erythroblasts. Sedimentation-enriched EBIs are still examined using traditional microscopy methods more than half a century after their discovery. Quantitative analysis is not afforded by these isolation procedures, thereby hindering precise determination of EBI counts and prevalence in the bone marrow and spleen. Quantification of cell aggregates co-expressing macrophage and erythroblast markers has been achieved using conventional flow cytometric techniques; nevertheless, the presence of EBIs within these aggregates remains an unanswered question, as visual confirmation of their EBI content is not permitted.