Flow Cytometry Applications
Flow Cytometry is a means of identifying and measuring certain physical and chemical characteristics of cells or particles as they travel in suspension. The benefit of flow cytometry is the rapid simultaneous measurement of several parameters on a cell by cell basis. Flow cytometry uses fluorescent probes to identify and characterize cells or particles. Cells or particles tagged with fluorescent molecules enter the cytometer via a fluid stream. The cells then pass by a laser, which emits a specific wavelength of light. The fluorescent probes are excited by the laser and then emit light. The fluorescent signal is detected and amplified, then translated into an electronic signal, which is sent to the computer. Information about the size and granularity of a cell is recorded, as well. The result is a visual presentation describing an individual or group of cellular events. The cells or particles can be separated by sorting, or the information can be collected and analyzed.
The research applications of flow cytometry include:
The most common application performed on the cytometer is immune-phenotyping. This technique identifies and quantifies populations of cells in a heterogeneous sample - usually blood, bone marrow or lymph. These cell subsets are measured by labeling population-specific proteins with a fluorescent tag on the cell surface. In clinical labs, immune-phenotyping is useful in diagnosing hematological malignancies such as lymphomas and leukemia.
Apoptosis, or programmed cell death, is a normal part of the life cycle of eukaryotic 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. 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.
Cell Proliferation Assays
Cell 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. The flow cytometer can measure proliferation by labeling resting cells with a cell membrane fluorescent dye, carboxyfluorescein succinimidyl ester (CFSE). When the cells are activated, they begin to proliferate and undergo mitosis. As the cells divide, half of the original dye is passed on to each daughter cell. By measuring the reduction of the fluorescence signal, researchers can calculate cellular activation and proliferation
Intracellular Calcium Flux
Cells interact with one another and their environment through signal transduction pathways. When these pathways are activated, membrane-bound calcium ion channels pump calcium into the cell and rapidly increase the intracellular calcium concentration. The higher calcium levels provide energy to the cell to respond to the external stimuli. The cytometer can monitor the flux of calcium into the cell and measure the extent to which cells respond to the stimuli.
Hoechst Side Population
Hoechst side population analysis is a valuable flow cytometry technique for the identification, enrichment, and purification of stem cells and early progenitors in a variety of human and non-human tissues. Although the technique was initially optimised on hematopoietic cells, it has since been used on different cell lines and tissues. read more...