See How to Schedule Instrument Use for scheduling details.See Flow Cytometry Fee Structure for details on fees.See Flow Cytometry Services for details about the sorters, analyzers, workstations and technical support available in the Flow Cytometry Laboratory. Flow cytometry is a technique used to measure properties of cells in a fluid as they pass through one or more laser beams.The cytometer can monitor the flux of calcium into the cell and measure the extent to which cells respond to the stimuli. The higher calcium levels provide energy to the cell to respond to the external stimuli. When these pathways are activated, membrane-bound calcium ion channels pump calcium into the cell and rapidly increase the intracellular calcium concentration. Intracellular Calcium FluxĬells interact with one another and their environment through signal transduction pathways. By measuring the reduction of the fluorescence signal, researchers can calculate cellular activation and proliferation. As the cells divide, half of the original dye is passed on to each daughter cell. When the cells are activated, they begin to proliferate and undergo mitosis. The flow cytometer can measure proliferation by labeling resting cells with a cell membrane fluorescent dye, carboxyfluorescein succinimidyl ester (CFSE). Cell Proliferation AssaysĬell proliferation assays are widely used in cell biology to measure cellular metabolic activity in response to stimuli such as growth factors, cytokines and other media components. These two distinct types of cell death, apoptosis and necrosis, can be distinguished by flow cytometry on the basis of differences in morphological, biochemical and molecular changes occurring in the dying cells. Cells die for a variety of reasons: through necrosis, brought on by external physical and chemical changes to the cell or through apoptosis, a process in which cells initiate a "suicide" program through internally controlled factors. ApoptosisĪpoptosis, or programmed cell death, is a normal part of the life cycle of eukaryotic cells. Along with determining cell cycle replication states, the assay can measure cell aneuploidy associated with chromosomal abnormalities. Cell Cycle Analysisįlow cytometry can analyze replication states using fluorescent dyes to measure the four distinct phases of the cell cycle. The sorter then uses sophisticated electronics and fluidics to identify and "kick" the cells of interest out of the fluidic stream into a test tube. The cytometer interrogates and characterizes each cell as it passes through the laser. The cell sorter is a specialized flow cytometer with the ability to physically isolate cells of interest into separate collection tubes. In clinical labs, immunophenotyping is useful in diagnosing hematological malignancies such as lymphomas and leukemia. These cell subsets are measured by labeling population-specific proteins with a fluorescent tag on the cell surface. This technique identifies and quantifies populations of cells in a heterogeneous sample - usually blood, bone marrow or lymph. Biopsies usually involve larger pieces of tissue than a cytology test needs, and a pathologist may examine. Because of this, cytology tests are usually painless. The most common application performed on the cytometer is immunophenotyping. By definition, cytology is the examination of individual cells or clusters of cells, so pathologists only need a very small sample to look at under a microscope for cytology tests. Additionally, information about cell size and internal structure (granularity of the cytoplasm, size of the cell nucleus and so on) is gained through light refraction and diffusion.Ī simultaneous measurement with different fluorescent dyes is possible, because the dyes used let themselves indeed be inspired by a combined but varied wavelength for the characteristic emission spectra of the respective dye.The research applications of flow cytometry include: Immunophenotyping The amount of photons emitted, which show a specific emission spectrum and can be detected by a photo detector, is proportional to the amount of marked molecules or antibodies per cell. After the laser pulse, the electrons fall back to their original state releasing energy in the form of photons. The marked cells, as single cell suspension, are analysed through the technique of the hydrodynamic focusing like pearls on a string that pass by a focused laser beam with appropriate wavelength.ĭuring stimulation of the fluorescence dye of the electrons through the monochromatic light of the laser beam, the molecules are lifted to a higher energy level. Therefore, the examined cells are generally marked with fluorescence dye-conjugated molecules (antibodies, receptors, streptavidin and so on). Molecules on the cell surface or inside the cell as well as proteins can be quantitatively determined with the help of flow cytometry.
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