Applications of Flow Cytometry
The research applications of flow cytometry include:
The most common application performed on the cytometer is immunophenotyping. 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, immunophenotyping is useful in diagnosing hematological malignancies such as lymphomas and leukemia.
The cell sorter is a specialized flow cytometer with the ability to physically isolate cells of interest into separate collection tubes. The cytometer interrogates and characterizes each cell as it passes through the laser. 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.
Cell Cycle Analysis
Flow cytometry can analyze replication states using fluorescent dyes to measure the four distinct phases of the cell cycle. Along with determining cell cycle replication states, the assay can measure cell aneuploidy associated with chromosomal abnormalities.
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.