Flow Cytometry
Flow cytometry enables the automated study of the physical and molecular properties of cells through the use of laser light [1]. The device used to perform this analysis is called a flow cytometer. An extension is the so-called FACS method (Fluorescence-activated Cell Sorting), in which the cells are not only measured but also sorted into different vials based on their fluorescence labeling [2].
The principle of flow cytometry is based on an antigen-antibody reaction, for which fluorescently labeled antibodies are used, or on direct staining of cells with dyes. For this purpose, the cells first flow individually by hydrodynamic focusing into a measuring chamber, the so-called flow cell [2]. Here, they are irradiated by a laser beam of a specific wavelength, which lifts the electrons of the fluorescent dye to a higher energy level. The electrons then fall back to their original level while emitting photons. A photodetector evaluates the resulting scattered light or fluorescence signal, which is proportional to the number of cells. Since each cell type produces a characteristic light scatter, this can also be used to make statements about the size and internal structure of the cells [3]. In addition to the detectors, a FACS instrument has an electrostatic sorting mechanism. Here, an electrode is used to reverse polarize a droplet from the sample stream at a cell to be sorted. A downstream electric field deflects the cell so that it falls into a different vessel than cells not to be sorted [4].
Principle of Fluorescence-activated Cell Sorting (FACS). The flow cytometer is loaded with the sample. In the measuring chamber, the cells are irradiated with a laser beam. Here, a detector measures the forward scatter light (FSC), which depends on the cell size, and the side scatter light (SSC), which is affected by the granularity of the cell. The droplets of the sample beam are polarized by an electrode and sorted according to their charge using an electric field (figure created with biorender.com).
Although the optical concept of a flow cytometer is very similar to that of a fluorescence microscope, the former can measure 50,000 to 100,000 cells in a few seconds, whereas a fluorescence microscope is not capable of such high-throughput screening. In addition, flow cytometry allows real-time measurement. The data obtained are usually presented in histograms or dotplots [4].
Flow cytometry is used primarily in basic medical and cell biology research. In the clinic, the method can be used in areas of hematology, infectiology, and immunology, while in biology it is used more for the qualitative and quantitative examination of cells. The method allows the analysis of cell functions as well as a distinction between, for example, living and dead cells [4]. In addition, flow cytometry is used in food analysis for the determination of the bacterial count in raw milk [5].
Do you want to learn more about flow cytometry? Then take a look at our blog! We also offer a wide range of high-quality antibodies that you can use for your next FACS examination:
Primary Antibodies for Flow Cytometry at Biomol
Secondary Antibodies for Flow Cytometry at Biomol
Sources
[1] Picot, J., Guerin, C.L., Le Van Kim, C. et al. Flow cytometry: retrospective, fundamentals and recent instrumentation. Cytotechnology. 64, 109–130 (2012).
[2] https://flexikon.doccheck.com/de/Durchflusszytometrie, 24.01.2023
[3] https://www.unimedizin-mainz.de/facs/durchflusszytometrie/prinzip-der-durchflusszytometrie.html, 24.01.2023
[4] https://de.wikipedia.org/wiki/Durchflusszytometrie, 24.01.2023
[5] Amtliche Sammlung von Untersuchungsmethoden gemäß § 64 LFGB: L 01.01-7:2002-05 Untersuchung von Lebensmitteln – Bestimmung der Keimzahl in Rohmilch – Durchflusszytometrische Zählung von Mikroorganismen (Routineverfahren). Beuth Verlag (2002).