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Flow Cytometry

Flow Cytometry is a powerful multi-parametric single cell analysis platform with wide applications in basic research and clinical medicine for the diagnosis, isolation and study of specific cell types and disease conditions.

Staining of human peripheral blood mononuclear cells (PBMC) using the T10B9 antibody (Cat #MAB1165).
T10B9 recognizes a unique monomorphic antigenic determinant of the T cell receptor expressed on the majorityh of human T cells. Human PMBC were prepared by density gradient separation and stained with T10B9 at the concentraiton of 10 μg/ml, with subsequent staining using goat anti-mouse light chain antibodies labeled with FITC (closed line histogram). The open line histogram represents an isotype (mouse lgM) control. Data courtesy of Dr. C. Keever-Taylor, Medical College of Wisconsin, Milwaukee, WI.

Flow cytometry is defined as the measurement of the cellular and fluorescent properties of particles in suspension as they pass by a laser or other light source. These particles are, in most cases, cells. The measurements are represented by changes in light scattered, light absorbed, and light emitted by a cell as it passes by fixed detectors directed off the light source. From these measurements, specific populations and subsets within a given cell population are defined and even isolated physically using a dedicated cell sorter typically by manipulating cell charge (see "Schematic Design of Flow Cytometry").

All light signals, whether from fluorescently labeled cells or from the beam scattered by cells, are transferred to a computer and transformed into digital signals. These signals are displayed in channels along a histogram graph. The typical output is a single parameter graph of light (typically fluorescence) intensity versus the number of cells (see the "T10B9 Staining" figure below) or two parameter histograms to distinguish subsets of cells.

Schematic Diagram of Flow Cytometry with sorting capacity. Cells are sorted according to their size and fluorescence.

Single parameter histograms display the relative fluorescence plotted against the number of events. The simplicity of this type of display is the main reason for its popularity. Its ease of use makes it ideal for simple assays, for instance distinguishing apoptotic cells from non-apoptotic cells (see the "Flow Cytometry Data" figure).

Schematic Diagram of Flow Cytometry with sorting capacity. Cells are sorted according to their size and fluorescence.

When one wishes to compare multiple parameters that are collected at the same time, more complex two-dimensional and even 3-dimensional diagrams are required. In these diagrams, one parameter is plotted against another in an X versus Y axis display. (see "Two Dimensional Histogram" figure).

Such diagrams or dot plots display the relationship between each of the two parameters. Individual subsets or cell populations within a sample are resolved based on the combined intensity levels of each of the two parameters. When comparing cell populations, subcellular debris or unimportant cell populations can be eliminated from the data by electronic gating. Gating involves using flow cytometry software to select a population within the graph that needs to be plotted. For instance, see figure "Gate for Target Population") we use gating to select the true population of single apoptotic and non-apoptotic cells from debris and aggregated cell populations. The gated cells are then displayed in a new graph using additional parameters to give measurable data (see figure "Processed Gated Population").

Log flourescence intensity profile of peripheral blood lymphocytes using FITC labeled CD4 (Cat. No. CBL127 ).

Two dimensional histogram, displaying a light scatter plot of white blood cells passing through a flow cytometer. X-axis shows forward light scatter indicative of size of cells, where as y-axis shows light scatter indicative of granularity of cells. Notice the thre distinctive populations of white blood cells.

Diagram shows the selection of the gate around target population excluding non-specific scatter. Diagram displays the flow cytometry data obtained when gated population is processed.

Labelling of cells in flow cytometry is often done using antibodies specific to particular subclasses of cells. Antibodies typically used are specific to external cell epitopes for live cell sorting. Internal epitope antibodies are typically only used on fixed cells that have first been permeabilized to allow the antibody penetration. Millipore has available numerous polyclonal and monoclonal antibodies specific to different cell types. Many of the groups of cell-type specific monoclonals have been given a cluster differentiation (CD) number by international convention. For example, the mono-are both excited with a 488 nm laser and two, their clonal antibodies recognizing epitopes of the antigen emission spectra are distinct with FITC at 530 nm site on helper T cells are known collectively as CD4 (green) and PE at 570-575 nm (orange). Advances (see "CD4 Labeling").

Sometimes, multiple CD markers are needed to identify one exact cell type or disease state. Lists of CD markers are available for cell identification in humans and other species.

Many antibodies used in flow cytometry are directly conjugated to a fluorochrome; however, many non-labelled primary antibodies are routinely used when combined with a labelled secondary antibody. There are two primary fluorochromes used in flow cytometry, fluoroisothiocyanate (FITC) and phycoerythrin (PE). The two key properties of these dyes that make them preferred tags are one, they are both excited with a 488 nm laser and two, their emission spectra are distinct with FITC at 530 nm (green) and PE at 570-575 nm (orange). Advances in fluorochrome chemistry and in flow cytometry instrumentation have now made multiple simultaneous cell labeling and sorting possible beyond the original two dyes (See fluorescence table in IHC section).

Because of its versatility and power, flow cytometry is routinely used in diagnostics, scientific research and pharmacology. The ability to sort cells by multiple parameters (simply limited by the number of electronic collectors, filters and available markers) makes flow cytometry an area of continuous growth, and one where antibodies and fluorescent markers combine into a truly functional and dynamic application.

T Cell Depletion of Human Bone Marrow using the T10B9 Monoclonal Antibody. T10B9 is a T cell specific antibody that selectively depletes cells bearing αβ+ T cell receptor using complement-mediated cytotoxicity (blue arrow in the upper pane), while sparing cells expressing -δ+ T cell receptor (lower panel). Human bone marrow suspensions were treated with0.35 μg/mL of Millipore's T1089, followed by two subsequent treatments with rabbit complement. Specific elimination of αβ+cells in T cell depletion protocols reduces risks of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation. The remaining -δ+ T cells presumably contribute to selective killing of host leukemia cells. Data courtesy of Dr. C. Keever-Taylor, Medical College of Wisconsin, Milwaukee, WI).

출처: http://www.millipore.com