Introduction
Cardiac fibroblasts are the main stromal cell type in the healthy myocardium. The activation of the third cardiac fibroblast has been implicated in the pathogenesis of heart disease. Probiotic stimuli activate fibroblast, which proliferates and differentiates into pathogenic myofibroblast that causes fibrotic phenotype in the heart. The production of type 1 collagen characterizes cardiac fibroblasts. Cytometric systems are high-through instruments that allow data collection in thousands of individuals within seconds.
The cytometric analysis is of two types:
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Flow cytometers.
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Slide-based cytometers.
The below article briefs about the simple flow cytometry method that helps in cardiology to isolate cardiac fibroblast from the murine heart.
What Is Flow Cytometry?
Flow cytometry is a fluorescence-based assay that enables the measurement of multiple characteristics, like protein abundance and population counts, from individual cells suspended in a solution. It is a powerful tool that enables quantitative, rapid, and accurate measurement of cell characteristics and provides unparalleled insight into the heterogenicity of cell populations. This is achieved using an instrument that directs a single stream of cells from a light source and measures the scatter and emission of light energy in various wavelengths. Fluorescent molecules with different excitation and emission characteristics can be combined, differentiated, and detected for multiple readouts from a single cell. These fluorescent molecules are attached to antibodies targeting specific proteins and protein modifications, and they can be dyes that are directly bonded to cellular components.
What Is Flow Cytometry Used For?
Flow cytometry is specifically used for several purposes. They are:
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Determining cell function.
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Determining cell characteristics.
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Cell sorting.
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Cell functioning.
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Detecting microorganisms.
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Finding biomarkers.
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Diagnosis and potential treatment of blood and bone marrow cancers.
What Are the Materials and Methods Used in Flow Cytometry in Cardiology?
The materials and methods used in flow cytometry in cardiology are:
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Isolation of Cardiac Cells: The cells are dissected to remove from the aorta and pulmonary artery and remaining ventricles and atria.
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Fibroblast Isolation: Fibroblasts are isolated from the human heart and skin tissue by an explant migrant. The fibroblast is then isolated by adding trypsin, followed by filtering through a 70-nanometer filter to allow a single fibroblast to pass through.
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Flow Cytometry:Dissociated cardiac cells are filtered through a cap in FACs tubes to ensure a single-cell suspension. The cells are treated with human FcR-blocking serum. Cells are incubated with secondary and primary antibodies and analyzed using a BD flow cytometer. Dual, single, and triple stain flow cytometry analyses are carried out.
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Immunohistochemistry and Immunocytochemistry: Human heart tissue is fixed in 4 % formaldehyde embedded in paraffin and 5-nanometer microtome section cuts. Antigen retrieval is performed before secondary and primary incubations.
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Antibodies for Flow Cytometry and Immunohistochemistry: Antibodies are selected to identify cardiomyocytes, smooth muscle cells, fibroblast, and endothelial cells based on current reports. Appropriate isotype control antibodies are used as negative controls for directly conjugated antibodies, and antibody control is used for non-conjugated antibodies.
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RT-PCR: The total RNA is isolated using TRIzol reagent from whole heart tissue and cell pellets of culture fibroblast. Polymerase chain reaction consists of initial denaturation of DNA followed by 40 cycles at 94-degree Celcius temperature.
What Are the Challenges and Limitations of Flow Cytometry?
Despite the advantages of flow cytometry, which offers cell biology and clinical applications, this technique has many downsides.
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The fluids in the system can be challenging, with cells or debris to maintain the consistency that causes blockades and changes in flow rates, which may affect the analysis.
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The risk is particularly when the cells are larger, like cancer cells.
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Contaminations in the piping can cause the system to block and flow to be interrupted. Laser alignment also ensures consistent results.
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Limitations include the potential of the cytometer that causes damage to cells, affecting analysis and subsequent culturing of cells, and the inability of single cells.
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The cost of the cytometers and the main high-parameter devices can be high, limiting the use of flow cytometry.
How Are Flow Cytometry Results Interpreted?
A pathologist interprets the flow cytometry results and places findings in a comprehensive laboratory report. The pathologist will consider the medical history, current symptoms, and the outcomes of the flow cytometry analysis.
A healthy cell shows a pattern of antigens that matches the type and maturity of the cell. These tests can help to prove the presence of lymphoma, leukemia, and other diseases. The abnormal results are found in the presence of:
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Chronic Lymphocytic Leukemia: A type of cancer of blood and bone marrow.
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Acute Lymphocytic Leukemia: Most commonly known as childhood cancer, that affects blood and bone marrow.
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Multiple Myeloma: Cancer that affects white blood cells called plasma cells.
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Non-Hodgkin's Lymphomas: A type of cancer that begins in the lymphatic system.
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Acute Myeloid Leukemia: It is a type of cancer that starts in the blood-forming cells of the bone marrow.
What Are the Future Developments in Flow Cytometry?
The main developments in the flow cytometry field have allowed a significant increase in the number of parameters with each sample and a reduction in the size of bulky machines. Future developments in the area will allow more extensive analyses on the border scale. Reducing the instrument size may allow easier placement in the laboratories and enable their use in different formats. Setting compensation, automatic pipetting, and allowing well plates to be analyzed automatically can increase the reliability of flow cytometry analyses.
Conclusion
Flow cytometry is a powerful tool that can work in multiple disciplines like virology, molecular biology, immunology, cancer biology, and other infectious diseases. Flow cytometry rapidly analyzes single cells or particles flowing past single or multiple lasers while suspended in a buffered salt-based solution. Particle is analyzed for visible light scattered in one or multiple fluorescence parameters. More sophisticated flow cytometry screening methods enable the evaluation of a wide array of blood types and blood-borne proteins combined with tools to inform iterative design improvements. Use of multiparametric flow cytometry design cycle and clinical use of the medical device contacts blood in infancy. Multiparametric flow cytometry enables the incorporation of functional outputs such as apoptosis, cell viability, reactive oxygen species, and cytokine production.
