What is cytometry?

Introduction

The flow cytometry allows for the examination of properties of individual particles (for instance, cells, tiny plastic spheres etc.) in a fluid stream.

The particle size as well as the granularity of the particles are among the physical properties that can be analysed. Furthermore, the fluorescence of the particles can also be analysed in different wavelengths. The term “FACS” is frequently used to express flow cytometry. “FACS” stands for Fluorescence Activated Cell Sorting and describes the separation of individual cells from the remaining cell suspension by means of analysed parameters. This is another application in the field of flow cytometry, in addition to pure cell analysis.

Principle

Molecules on the cell surface or inside the cell as well as proteins can be quantitatively determined with the help of flow cytometry.

Therefore, the examined cells are generally marked with fluorescence dye-conjugated molecules (antibodies, receptors, streptavidin and so on).

The marked cells, as single cell suspension, are analysed through the technique of the hydrodynamic focusing like pearls on a string that pass by a focused laser beam with appropriate wavelength.

During stimulation of the fluorescence dye of the electrons through the monochromatic light of the laser beam, the molecules are lifted to a higher energy level. After the laser pulse, the electrons fall back to their original state releasing energy in the form of photons. The amount of photons emitted, which show a specific emission spectrum and can be detected by a photo detector, is proportional to the amount of marked molecules or antibodies per cell. Additionally, information about cell size and internal structure (granularity of the cytoplasm, size of the cell nucleus and so on) is gained through light refraction and diffusion.

A simultaneous measurement with different fluorescent dyes is possible, because the dyes used let themselves indeed be inspired by a combined but varied wavelength for the characteristic emission spectra of the respective dye.

Common applications

• Determination of DNA content through a DNA-Intercalator (e.g. Propidium iodide) for analysis of the cell cycle phases or of the subG1-fraction (Marker for Apoptosis).
•  AnnexinV/PI-double staining to discriminate apoptotic and necrotic cells.
• Quantification of the surface proteins in cells of the blood system with the help of fluorochrome-labelled antibodies.
• Analysis of the GFP-Expression (e.g. to determine transfection efficiency or the transcription levels of particular genes)
• Sorting out single cell populations from complex cell suspensions (e.g.blood) Sortieren einzelner Zellpopulationen aus komplexen Zellsuspensionen (z.B. Blut) by means of analysed parameters.
• And various others...

  

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