The importance of Foxp3 antibody and fixation/permeabilization buffer combinations in identifying CD4+CD25+Foxp3+ regulatory T cells
Open accessAbstract
Foxp3 is a key marker for CD4+ regulatory T cells (T regs) and was used in developing a multiparameter flow cytometric panel to identify T regs. Achieving reproducible staining and analysis first required optimization of Foxp3 staining. We present a comparative study of PCH101, 236A/E7, 3G3, 206D, 150D, and 259D/C7 clones of anti‐human‐Foxp3 antibodies used in combination with five different fixation/permeabilization buffers. Staining for CD25, CD152, and CD127 was also compared between fixation/permeabilization treatments. Promising antibody/buffer combinations were tested in a panel of peripheral blood mononuclear cells from 10 individuals, and then on fresh versus frozen cells from four individuals. Finally, different fluorochromes coupled to two representative antibodies were compared to optimize separation of Foxp3+ from Foxp3− events. Foxp3 gates were set using two gating strategies based on CD127+CD25− “non‐T regs” or based on isotype controls. For Foxp3 staining, the best conditions for fixation/permeabilization were obtained using the eBioscience Foxp3, Imgenex, BioLegend, and BD Foxp3 buffers. Comparing results from 10 subjects, 259D/C7, PCH101, 236A/E7, and 206D antibodies yielded statistically higher levels of Foxp3 cells than those by 150D and 3G3 antibodies (mean = 6.9, 5.1, 4.7, and 3.7% compared with 1.7, and 0.3% of CD25+Foxp3+ events within CD4+ cells, respectively). Importantly, the “nonspecificity” of some antibodies observed with a Foxp3 gate based on isotype controls could be eliminated by setting the Foxp3 gate on “non‐T regs”. Better separation of Foxp3+ and Foxp3− populations was observed using the PCH101 clone coupled to Alexa647 compared with FITC or the 259D/C7 clone coupled to PE compared with Alexa488 fluorochrome. Foxp3 staining can be highly variable and depends on the choice of antibody/buffer pair and the fluorochrome used. Selecting the correct population for setting the Foxp3 gate is critical to avoid including non‐T regs in the Foxp3+ gate. The experiments presented here will aid in optimization of flow cytometry staining panels to quantify T reg frequencies in humans. © 2009 International Society for Advancement of Cytometry
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There is a general consensus that the 236A/E7 and 259D anti-human FOXP3 Ab clones are optimal.
“Several studies have compared different anti-human FOXP3 Ab clones, and although there is some debate, there is a general consensus that the 236A/E7 and 259D clones are optimal [394-398].”
From Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition) (2021) · cites this paper as [396]
The Discussion (page unavailable) identifies 259D/C7 and 236A/E7 among four clones that yielded the most distinct putative regulatory T-cell populations. It does not establish a general consensus that those two alone are optimal; it also names PCH101 and 206D, and reports that staining depends on the buffer and fluorochrome used. (LLM, conf=0.86)
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- Authors
- Law, J. P., Hirschkorn, D. F., Owen, R. E., Biswas, H. H., Norris, P. J., & Lanteri, M. C.
- Affiliations
- Blood Systems Research Institute (US)
- University of California, San Francisco (US)
- Corresponding author
- Lanteri, Marion C.
- Venue
- Cytometry Part A
- Publisher
- Wiley
- Published
- 2009-10-20
- Volume
- 75A
- Issue
- 12
- Pages
- 1040-1050
- ISSN
- 1552-4922 (ISSN-L)
- 1552-4930
- PMC
- PMC2862733
- OpenAlex
- W1966941710
- Cited by
- 71
- References
- 33
- Citations by year
- 2024: 3 · 2023: 7 · 2022: 1 · 2021: 4 · 2020: 2 · 2019: 6 · 2018: 3 · 2017: 2 · 2016: 7 · 2015: 5 · 2014: 5 · 2013: 11 · 2012: 5
- FWCI
- 2.16
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Cytometry Part A (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Heart, Lung, and Blood Institute (HL083388-01, R01HL083388)
- Topics
- Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
- Life Sciences › Immunology and Microbiology › Immunology › Monoclonal and Polyclonal Antibodies Research
- Life Sciences › Immunology and Microbiology › Immunology › Psoriasis: Treatment and Pathogenesis
- Keywords
- Foxp3, regulatory T cells, CD4+CD25+ T cells, antibody clones, CD127, CD25, CTLA-4, peripheral blood mononuclear cells
- MeSH terms
- Antibodies, Buffers, Humans, Permeability, Staining and Labeling, CD4 Antigens, Tissue Fixation, T-Lymphocytes, Regulatory, Forkhead Transcription Factors, Interleukin-2 Receptor alpha Subunit
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