The DOI matches its Crossref record; 18 of the 18 references it cites were found in a registry or an index.
Brefeldin A, but not monensin, completely blocks CD69 expression on mouse lymphocytes:
Abstract
Flow cytometry is increasingly used for cytokine detection where it serves to complement ELISA (enzyme-linked immunosorbent assay) and ELISPOT assays. Since it is possible to stain both extracellular epitopes and intracellular cytokines on the same cells, this is a powerful technique for analysing cytokine expression in defined cell populations. However unstimulated cells do not express cytokines. Thus, appropriate stimulation is a prerequisite for studying cytokine expression. Here phorbol 12-myristate 13-acetate (PMA)/ionomycin in vitro stimulation has been applied. In order to accumulate the cytokines within the cells, protein secretion needs to be inhibited, by the addition of reagents that inhibit protein secretion during the stimulation. The two most widely used reagents are monensin and brefeldin A (BFA). These reagents differ somewhat in their mode of action, which might explain their different effects. Monensin is an inhibitor of trans-Golgi function, while BFA inhibits protein transport between the endoplasmic reticulum (ER) and the Golgi. CD69, a very early activation marker on lymphocytes and neutrophils, was monitored in order to measure the efficacy of the protein secretion inhibition. Here we report that: (a) BFA, but not Monensin, is able to completely block extracellular CD69 expression on mice splenocytes after in vitro stimulation with PMA/ionomycin; (b) Monensin is more toxic than BFA and increases the relative amount of CD4+ cells due to a more profound increase in dead cells in the CD4- population; (c) CD69 is a useful marker when setting up intracellular staining of cytokines for flow cytometry.
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“BFA will help to maximize the capture of TNF-α, IFN-γ, and IL-17A, but blocks the surface expression of some molecules, such as the T-cell activation marker CD69 [162].”
From Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition) (2021) · cites this paper as [162]
Citations
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Cited by
Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)
Cossarizza, A., Chang, H., Radbruch, A., Abrignani, S., Addo, R., Akdis, M., Andrä, I., Andreata, F., Annunziato, F., Arranz, E., et al. (2021) · European Journal of Immunology · doi:10.1002/eji.202170126 · Cited by 531
Metadata
- Authors
- Nylander, S., & Kalies, I.
- Corresponding author
- Nylander, Sven
- ORCID
- Nylander, Sven
- Venue
- Journal of Immunological Methods
- Publisher
- Elsevier BV
- Published
- 1999-04
- Volume
- 224
- Issue
- 1-2
- Pages
- 69-76
- ISSN
- 0022-1759 (ISSN-L)
- OpenAlex
- W4752647
- Cited by
- 91
- References
- 18
- Citations by year
- 2026: 3 · 2025: 2 · 2024: 1 · 2023: 5 · 2022: 2 · 2021: 7 · 2020: 1 · 2019: 3 · 2017: 2 · 2016: 4 · 2015: 1 · 2014: 2 · 2013: 2 · 2012: 4
- FWCI
- 2.12
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Immunological Methods (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
- Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
- Life Sciences › Immunology and Microbiology › Immunology › Immunotoxicology and immune responses
- Keywords
- brefeldin A, monensin, CD69, lymphocyte activation markers, protein secretion inhibition, intracellular cytokine staining, PMA/ionomycin stimulation, splenocytes, CD4+ T cells, endoplasmic reticulum to Golgi transport, reagent toxicity
- MeSH terms
- CD69 Antigens, Animals, Antigens, Differentiation, T-Lymphocyte, Cells, Cultured, Female, Flow Cytometry, Intracellular Fluid, Lymphocytes, Mice, Inbred BALB C, Monensin, Protein Synthesis Inhibitors, Spleen, Staining and Labeling, Antigens, CD, Gene Expression, Cytokines, Brefeldin A, Lectins, C-Type, Mice
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