Antibodies in Flow Cytometry

Flow cytometry is an invaluable tool used to analyze the chemical and physical properties of cells. This laboratory technique uses an antibody conjugated with a fluorochrome for cell analysis.


Antibodies. Image Credit: Corona Borealis Studio/

The development of a specialized flow cytometer in the 1970s, triggered by the HIV pandemic, paved the way to a broad range of applications such as protein modification studies, immunophenotyping, and analysis of intracellular antigens. The most important clinical application of flow cytometry is in hematologic malignancy diagnosis.

Principles of flow cytometry

In flow cytometric analysis, suspension of a single cell or particle is prepared and aspirated into a flow cell. The particles are made to pass through a focused laser beam one at a time and the light is either absorbed or scattered by the cells. Antibodies labeled with fluorochromes are attached to the cell surface, which helps the cells re-emit absorbed light as fluorescence.

The fluorescence signals are received by an array of photodiodes and amplified. The electrical pulses that are formed are converted into digital data that can be analyzed, displayed, and stored in a computer. Thus, statistically valid quantitative data related to a huge number of cells can be obtained in a very short period of time.

Role of antibodies in flow cytometry

Antibodies are an invaluable component of flow cytometry. The advent of monoclonal antibodies in 1977 promised an unlimited supply of highly specific antibodies and dramatically changed the flow cytometry technique. Monoclonal antibodies are produced from single B-cell clones developed in hybridoma cells.

They have homogeneous antigen-binding sites and therefore, are highly specific to antigenic determinants. Over the years, specific monoclonal antibodies for murine MHC antigens and murine/rat helper T cells have been developed. The identification of 3 murine monoclonal antibodies that are specific for human T-cells - OKTl, OKT3, and OKT-l-lI7 – opened up new avenues for human studies.

Monoclonal antibodies generated against a wide range of biological molecules such as glycoproteins, proteins, carbohydrates, glycolipids, histones, lysosomes, and cytokines have been produced over the years.

The development of monoclonal antibodies against specific phosphoepitopes that can help in the detection of protein activation states has enabled the use of flow cytometry to study cellular function. Since antibodies can recognize and bind to antigenic epitopes, they can be used to identify different molecules with specific epitopes.

The size and shape of the antibody used and its conjugates influence the staining measurements in flow cytometry, especially in the case of cytoplasmic or intranuclear staining. Controlled permeabilization is crucial to facilitate the penetration of the fluorochrome-antibody conjugate into the nucleus or the cytoplasm and to achieve optimum extracellular staining. Also, high concentrations of antibodies in flow cytometry can lead to non-specific binding which will complicate the process.

Antibody stability is another concern that affects staining in flow cytometry. Exposure to extreme salt concentration or pH decreases the stability of antibodies and they tend to form soluble aggregates and precipitated polymers.

This is probably because of their increased hydrophobicity, which increases the possibility of non-specific binding. Therefore, these aggregates and polymers need to be removed from antibodies before using them in flow cytometry. All these factors need to be carefully considered before the formulation of antibody-conjugates for flow cytometry.

Sigma® Life Science, the Life Science division of Sigma-Aldrich, provides monoclonal antibodies validated specifically for flow cytometry in pure form or conjugated to some commonly used fluorochromes. The company offers antibodies against MHC and related antigens, immunoglobulins, and CD and related antigens.

Thermo Fisher Scientific offers antibodies conjugated to 24 different fluorescent dyes and proteins for use in flow cytometry. This allows more flexibility in experiment design and expands the range of the detectable parameter of flow cytometry.


Further Reading

Last Updated: Jul 1, 2022

Susha Cheriyedath

Written by

Susha Cheriyedath

Susha is a scientific communication professional holding a Master's degree in Biochemistry, with expertise in Microbiology, Physiology, Biotechnology, and Nutrition. After a two-year tenure as a lecturer from 2000 to 2002, where she mentored undergraduates studying Biochemistry, she transitioned into editorial roles within scientific publishing. She has accumulated nearly two decades of experience in medical communication, assuming diverse roles in research, writing, editing, and editorial management.


Please use one of the following formats to cite this article in your essay, paper or report:

  • APA

    Cheriyedath, Susha. (2022, July 01). Antibodies in Flow Cytometry. AZoLifeSciences. Retrieved on June 15, 2024 from

  • MLA

    Cheriyedath, Susha. "Antibodies in Flow Cytometry". AZoLifeSciences. 15 June 2024. <>.

  • Chicago

    Cheriyedath, Susha. "Antibodies in Flow Cytometry". AZoLifeSciences. (accessed June 15, 2024).

  • Harvard

    Cheriyedath, Susha. 2022. Antibodies in Flow Cytometry. AZoLifeSciences, viewed 15 June 2024,


The opinions expressed here are the views of the writer and do not necessarily reflect the views and opinions of AZoLifeSciences.
Post a new comment

While we only use edited and approved content for Azthena answers, it may on occasions provide incorrect responses. Please confirm any data provided with the related suppliers or authors. We do not provide medical advice, if you search for medical information you must always consult a medical professional before acting on any information provided.

Your questions, but not your email details will be shared with OpenAI and retained for 30 days in accordance with their privacy principles.

Please do not ask questions that use sensitive or confidential information.

Read the full Terms & Conditions.

You might also like...
Researchers Develop Strategy to Replace Diseased Blood System Affected by Leukemia