Flow cytometry
is a powerful application widely used across immunology, molecular biology, microbiology, cancer biology and infectious disease monitoring.
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Antibodies are important reagents that not only drive the detection capabilities of flow cytometry, but also facilitate a great deal of flexibility in experimental design.
These protein-based reagents are responsible for detecting specific target antigens, both surface and intracellular, that make it possible to analyze and sort cells, and study cellular states, processes, and phenotypes.
This page offers a guide to help navigate and select
antibodies from a vast catalog
across different suppliers.
Select antibodies validated for flow cytometry
One of the first attributes users should look for when choosing antibodies is validation for the flow cytometry application.
Validation is proof an antibody recognizes its specific target and will work reliably for a specific application.
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As protein reagents, antibodies are very sensitive and specific to the epitope conformation of its target immunogen.
For instance, antibodies are either fit for binding to unfolded target proteins, or properly folded native proteins.
Antibodies that are effective for denatured proteins in Western blotting may not perform as well for folded proteins in flow cytometry.
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Responsible suppliers will provide validation data in the product datasheet, which serves as a useful resource for prospective buyers.
Users should review such data carefully and also look out for several key pieces of information.
The cell model used in validation should ideally be applicable to the intended experiments.
A recommended protocol, if any, can serve as a guide to ensure the best antibody performance.
For added security, also look for of any potential off-target effects.
After procuring antibodies, it is also good practice to perform in-house validation to optimize for the user's own cells and instrument.
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Learn more about flow cytometry antibody validation.
Consider directly conjugated primary antibodies
Using directly conjugated primary antibodies, which omits the need to use secondary antibodies, simplifies the staining process.
A simpler sample preparation protocol can reduce the potential for mistakes.
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This also increases the pool of antibodies to choose from.
As users will no longer need to worry about the host species of the antibody, multiple antibodies raised in the same species can be used, allowing greater flexibility.
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Careful planning of a multicolor flow cytometry experiment is crucial to ensure robust resolution, sensitivity, and biological accuracy.
Our tool lets you design your flow panel using instrument configurations based on any flow cytometer of your choice.
Monoclonal versus polyclonal antibodies
Polyclonal antibodies, which can bind multiple epitopes for a given target antigen, are known for generating good immunodetection signals.
However, this comes with an increased risk for cross-reacting with proteins other than the bodies target.
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If cross-reactivity is anticipated to be an important issue, monoclonal antibodies should be used.
Monoclonal antibodies are homogenous, recognize only one epitope and exhibit less batch-to-batch variability.
They have also been used to define specific antigens that have since received “cluster of differentiation,” or “CD,” designations.
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Users can also consider using recombinantly produced monoclonal antibodies for greater accuracy and reproducibility.
Recombinant antibodies are touted for offering low off-target binding and the highest batch-to-batch consistency.
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Certain recombinant antibodies are also engineered for low Fc receptor binding, minimizing non-specific staining.
Isotype control antibodies
Immunostaining is subject to unwanted background that can arise from artifacts, debris, and nonspecific binding to cellular components.
Certain antibody-binding immune cells express Fc receptors, which can bind experimental antibodies and cause non-specific staining.
Flow cytometry experiments incorporating these cell types should include isotype control antibodies as negative controls to account for such non-epitope-driven binding.
The isotype control is similar to the primary antibody, but is raised against an antigen not present in the experimental sample.
The control should also match the host species, antibody class, and subclass, and conjugate of the primary antibody.
Learn more about isotype controls here.
References
1. McKinnon KM. Flow Cytometry: An Overview. Curr Protoc Immunol. 2018;120:5.1.1-5.1.11. Published 2018 Feb 21. doi:10.1002/cpim.40
2. Easthope E. Antibody Validation for Flow Cytometry. Biocompare. 2021 Nov 23 [cited 2024 Jun]. Available from: https://www.biocompare.com/Editorial-Articles/579837-Antibody-Validation-for-Flow-Cytometry/
3. Voskuil J, Voskuil-Schlagwein A. 10 Commandments for Saving Money in the Research Antibody Industry. Biocompare. 2022 Sep 28 [cited 2024 Jun]. Available from: https://www.biocompare.com/Editorial-Articles/590327-10-Commandments-for-Saving-Money-in-the-Research-Antibody-Industry/
4. Easthope E. Selecting the Right Antibody for Flow Cytometry. Biocompare. 2019 Aug 12 [cited 2024 Jun]. Available from: https://www.biocompare.com/Editorial-Articles/362217-Selecting-the-Right-Antibody-for-Flow-Cytometry/
5. Easthope E. Immunophenotyping by Flow Cytometry. Biocompare. 2023 Jul 11 [cited 2024 Jun]. Available from: https://www.biocompare.com/Editorial-Articles/597499-Immunophenotyping-by-Flow-Cytometry/
Recommended Reading:
This article looks at the basic principles of flow cytometry and key steps in the sample preparation workflow, and suggests ways to improve the quality of your data.
This article explores what’s new in the flow cytometry space, where novel reagents, antibodies, and instrumentation are expanding flow’s capabilities and making the technology more accessible.