#FACS ANALYSIS SERIES#

Flow cytometry data will plot each event independently, and will represent the signal intensity of light detected in each channel for every event.įlow cytometry data is typically represented in one of two ways: histograms, which measure or compare only a single parameter, and dot-plots which compare 2 or 3 parameters simultaneously on a two- or three-dimensional scatter-plot.Ī histogram typically plots the intensity detected in a single channel along one axis and the number of events detected at that intensity is in a separate axis. In a flow cytometry experiment, every cell that passes through the flow cytometer and is detected will be classified as a distinct event.Īdditionally, each type of light that is detected by the flow cytometer (forward-scatter, side-scatter, and each wavelength of fluorescence emission) will be assigned its own unique channel. Upon exiting the flow chamber, electromagnets will sort cells by charge into separate vessels.Based on these parameters, the FACS machine uses an electrode to impose an electrical charge on each cell.User-defined parameters provide information on how cells should be sorted.Forward-scatter, side-scatter, and fluorescent data is collected, as in conventional flow cytometry.Using FACS a researcher can physically sort a heterogeneous mixture of cells into different populations.īy using highly specific antibodies tagged with fluorescent dyes, a researcher can perform FACS analysis and simultaneously gather data on, and sort a sample by a nearly limitless number of different parameters. In practice, there are differences between the two methods.įACS is a derivative of flow cytometry that adds an exceptional degree of functionality. The terms flow cytometry and fluorescence-activated cell sorting (FACS) are often used interchangeably. FACS: Sorting Cells based on Flow Cytometry Data In this instance, neutrophils could be selectively labeled by virtue of their expression a neutrophil specific marker like ELANE. However, three classes of granulocytes (neutrophils, basophils, and eosinophils) are very similar in size and structure, giving them similar light-scattering properties. For example, in the plot above, a researcher may be able to distinguish between granulocytes and lymphocytes using forward and side-scattered light. While some identities can be confirmed by forward and side-scatter profiles, labeling with a cell-type specific marker always provides greater resolution and certainty when profiling complex heterogeneous populations of cells. These cells generate high forward- and side-scatter signals. These cells generate a medium forward-scatter and low side-scatter signal intensity G Probable granulocytes, large cells with high internal complexity/granularity. L/M Probable leukocytes/monocytes, small to medium cells with low internal complexity/granularity. Cell populations are marked by their probable identity: D Presumed debris, very small items with low low forward- and side- scatter. Image: A mock flow cytometry dot-plot, plotting forward vs side-scattered from a population of leukocytes. Data acquired by the sensors is compiled and integrated to build a comprehensive picture of the sample.įACS Antibodies currently used for ResearchĪ mock flow cytometry dot-plot, plotting forward vs side-scattered from a population of leukocytes.Ĭell populations are marked by their probable identity.

#FACS ANALYSIS SERIES#

A series of sensors detect the types of light that are refracted or emitted from the cells.Light is used to illuminate the cells in the channel.Sample cells are passed through a narrow channel one at a time.clusters of differentiation or CD markers) can be used to better identify and segregate specific sub-populations within a larger group. When additional information is required, antibodies tagged with fluorescent dyes, and raised against highly specific cell surface antigens (e.g. For example, in immunology flow cytometry is used to identify, separate, and characterize various immune cell subtypes by virtue of their size and morphology. Flow cytometry is a particularly powerful method because it allows a researcher to rapidly, accurately, and simply collect data related to many parameters from a heterogeneous fluid mixture containing live cells.įlow cytometry is used extensively throughout the life and biomedical sciences, and can be applied in any scenario where a researcher needs to rapidly profile a large population of loose cells in a liquid media. Originally developed in the late 1960s, flow cytometry is a popular analytical cell-biology technique that utilizes light to count and profile cells in a heterogenous fluid mixture. Custom Recombinant Antibody (rAbs) Services.Annexin V-FITC Apoptosis Detection Kits.

Facs analysis