Flow cytometry is a laser based technology that allows for simultaneous multiparametric analysis of physical and/or chemical characteristics of cells at the single-cell level. Fluorescence-labeled cells are carried to sensing area in sheath fluid and cross laser beam one a time. Information about the properties of cells, including cell size, granularity by scattered light and relative fluorescence intensity, are transmitted to light detectors after passing through a series of optical filters. This information is then converted into digital electronic signals and collected by specialized computer programs for further analysis
Flow cytometry technique can be used in a wide range of research applications such as
• simultaneous analysis of levels of surface and intracellular markers (e.g. intracellular cytokine level)
• cell viability, apoptosis and proliferation,
• DNA content and cell cycle analysis,
• gene expression and transfer (e.g. transfection efficiency),
• intracellular Calcium flux
• activation states and oxidative burst
Fluorescence-activated cell-sorting (FACS)
Fluorescence-activated cell sorting is a specialized type of flow cytometry where a heterogeneous
mixture of cell suspension can be sorted into up to four populations of interest based on specific light scattering and fluorescent characteristics. Cells are sorted one a time into single-cell-containing droplets that are broken off from the stream by a vibrating mechanism. As droplets flow pass laser beam and light detectors, those with cell of interest that meets the pre-set criteria are placed with an electrical charge and deflected to different angles before entering designated collection tubes.
FACS can be used for
• purification of rare populations such as cancer stem cells
• isolation of particular cell subpopulations of interest (e.g. GFP-positive transfected cells, lineage negative bone marrow cells by surface marker staining)
• establishment of monoclonal transfected cell line by single cell isolation.