Flow cytometry data analysis pdf

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Get access to the best antibodies, discovery platforms, and know-how to advance your diagnostic and therapeutic programs. Flow cytometry is a popular laser-based technology to analyze the characteristics of cells or particles. Discover more with our introduction to flow cytometry. Flow cytometry is a widely used method for analyzing the expression of cell surface and intracellular molecules, characterizing and defining different cell types in a heterogeneous cell population, assessing the purity of isolated subpopulations and analyzing cell size and volume. It allows simultaneous multi-parameter analysis of single cells. It is predominantly used to measure fluorescence intensity produced by fluorescent-labeled antibodies detecting proteins, or ligands that bind to specific cell-associated molecules such as propidium iodide binding to DNA. The staining procedure involves making a single-cell suspension from cell culture or tissue samples.

The cells are then incubated in tubes or microtiter plates with unlabeled or fluorochrome-labeled antibodies and analyzed on the flow cytometer. Sheath fluid focuses the cell suspension, causing cells to pass through a laser beam one cell at a time. Forward and side scattered light is detected, as well as fluorescence emitted from stained cells. When a cell suspension is run through the cytometer, sheath fluid is used to hydrodynamically focus the cell suspension through a small nozzle. Light scattered from the cells or particles is detected as they go through the laser beam. Fluorescence detectors measure the fluorescence emitted from positively stained cells or particles. Cells or particles passing through the beam scatter light, which is detected as FS and SS.

FS correlates with cell size and SS is proportional to the granularity of the cells. In this manner, cell populations can often be distinguished based on differences in their size and granularity alone. Light scatter as the green laser interrogates the cell. The direction of light scattered by the cell correlates to cell size and granularity. A useful example of this is when running blood samples on the flow cytometer. Larger and more granular granulocyte cells produce a large population with high SS and FS.

Monocytes are large cells, but not so granular, so these produce a separate population with high FS but lower SS. Smaller lymphocytes and lymphoblasts produce a separate population with less FS. They are not granular cells, so also have low SS. Therefore, these cells can be separated into different populations based on their FS and SS alone. Dot plot of FS versus SS.

Each dot represents a single cell analyzed by the flow cytometer. The characteristic position of different cell populations is determined by differences in cell size and granularity. As well as separating cells based on FS and SS, cells can also be separated by whether they express a particular protein. In this case, a fluorochrome is often used to stain the protein of interest. Fluorochromes used for the detection of target proteins emit light when excited by a laser with the corresponding excitation wavelength. These fluorescent stained cells or particles can be detected individually.

Forward and side scattered light and fluorescence from stained cells are split into defined wavelengths and channeled by a set of filters and mirrors within the flow cytometer. The fluorescent light is filtered so that each sensor will detect fluorescence only at a specified wavelength. Fluorescent light is filtered so that each PMT detects a specific wavelength. PMT will detect light emitted from FITC at a wavelength of approximately 519 nm. Each PMT will also detect any other fluorochromes emitting at a similar wavelength to the fluorochrome it is detecting. These are detected by the PMT and converted to a voltage pulse, known as an event.