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Overview of Flow Cytometry

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Flow cytometry is a multiplex technique that allows for the simultaneous identification or phenotyping of multiple cell populations in a single sample. Both cell surface and intracellular proteins can be identified using this technique, which is based on the binding of fluorescently-conjugated antibodies to target antigens.


What is flow cytometry?


Flow cytometry is a semi-quantitative technique that allows for identification of cell populations from single cell suspensions of cultured or primary cells. Various cell populations can be distinguished by the relative increase in expression of specific markers over an unstained negative control sample. Because of its multiplex capabilities, flow cytometry is powerful in its ability to identify multiple cell populations from a single sample.


How does it work?


Flow cytometry uses fluorescently-tagged antibodies to recognize proteins of interest on target cells. First, cells are isolated from culture or from primary tissue and suspended in a buffer solution. To block non-specific binding of antibodies to these cells during the staining step, the Fc receptors are blocked using an anti-CD16/32 antibody. Next, the cells are incubated with the desired cocktail of antibodies to cell surface antigens. If staining for intracellular or nuclear antigens, the cells are subsequently permeablized, and antibodies to those proteins are added1. Cell proliferation can also be measured by the addition of CFSE or a cell trace dye whose fluorescence intensity is serially diluted with each cell division2. Finally, cells are fixed in a paraformaldehyde-containing solution before being read on a cytometer.

As cells are pulled through the cytometer, they cross lasers of different wavelengths, which activate the fluorophores now bound to each cell. The cytometer detects the amount of fluorescence emitted by each cell in each channel. When compared to unstained cells, this read-out provides the relative positivity of each cell for each targeted protein. Using the data, cell populations can be identified and their relative increase or decrease in frequency following stimulation can be quantified.


Why flow cytometry?


The field of flow cytometry is rapidly growing with new adaptations of the technique constantly being developed. The most common flow cytometric technique binds fluorescently-conjugated antibodies to target proteins on the cell surface or intracellularly. The read-out provides the staining intensity of each antibody on each cell, allowing researchers to identify cell populations based on positive or negative staining for a set of cell type-specific markers. One adaptation of this technique combines flow cytometry with immunofluorescent imaging, giving a photograph of each cell as it passes through the cytometer. This adds information on the localization of each protein within the cell, and provides insight into the dynamics of protein dynamics and interactions3. Another extension of the technique, mass cytometry, uses antibodies conjugated to heavy metal ions instead of to fluorochromes4. Because there is less spillover between heavy metal conjugates, mass cytometry can identify many more proteins simultaneously than can traditional flow cytometry.


Bethyl Laboratories sells high quality antibodies for use in flow cytometry. For example, these products have been used recently to:

  • Elucidate mechanisms underlying maintenance of embryonic stem cells5
  • Identify proteins involved in the progression of sickle cell disease6
  • Understand the development of adult neurodegenerative disorders7
  • Characterize DNA-binding proteins involved in aging8


Below is the entire list of targets involved in flow cytometry. Can’t find what you are looking for? Bethyl offers a custom antibody service.




1. Freer G, Rindi L. 2013. Intracellular cytokine detection by fluorescence-activated flow cytometry: basic principles and recent advances. Methods. May 15;61(1):30-38.

2. Lyons AB, Blake SJ, Doherty KV. 2013. Flow cytometric analysis of cell division by dilution of CFSE and related dyes. Curr Protoc. Cytom. Chapter 9: Unit 9.11.

3. McFarlin BK, Gary MA. 2017. Flow cytometry what you see matters: Enhanced clinical detection using image-based flow cytometry. Methods. Jan 1;112:1-8.

4. Spitzer MH, Nolan GP. 2016. Mass Cytometry: Single Cells, Many Features. Cell. May 5;165(4):780-791.

5. Fagnocchi L, Cherubini A, Hatsuda H, Fasciani A, Mazzoleni S, Poli V, Berno V, Rossi RL, Reinbold R, Endele M, et al. 2016. A Myc-driven self-reinforcing regulatory network maintains mouse embryonic stem cell identity. Nat Commun. June 15;7:11903.

6. Costa FC, Fedosyuk H, Chazelle AM, Neades RY, Peterson KR. 2012. Mi2β is required for γ-globin gene silencing: temporal assembly of a GATA-1-FOG-1-Mi2 repressor complex in β-YAC transgenic mice.. PLoS Genet. 8(12):e1003155.

7. Toulouse A, Au-Yeung F, Gaspar C, Roussel J, Dion P, Rouleau GA. 2005. Ribosomal frameshifting on MJD-1 transcripts with long CAG tracts. Human Molecular Genet. Sept 15;14(18): 2649-2660.

8. Schuler N, Rube CE. 2013. Accumulation of DNA damage-induced chromatin alterations in tissue-specific stem cells: the driving force of aging? PLoS One. May 17;8(5):e63932.