Intracellular cytokine optimization and standard operating procedure
Abstract
We describe here a method for optimizing the use of polychromatic flow cytometry (with up to 17 fluorochromes simultaneously) in surface and intracellular staining of human T lymphocytes. We will highlight and discuss how to procedurally optimize key steps in the experimental process before an intracellular cytokine staining assay protocol is finalized. These include but are not limited to the titration of monoclonal antibodies, use of a dead-cell discriminator and 'dump' channel, selection of a cytokine secretion inhibitor, selection of fixation and permeabilization reagents, and inclusion of compensation controls. Building on this basic protocol, we then establish a polychromatic assay designed to detect five separate functions of T lymphocytes (production of three cytokines and one chemokine, and degranulation) while simultaneously identifying multiple surface markers on the responding cells.
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Appropriately titrated antibodies should be used, and this concentration is usually not the concentration suggested by the supplier.
“always use appropriately titrated Abs, which is usually not the concentration suggested by the supplier. The ins and outs of titrating Abs can be found in the publication of Lamoreaux et al. [174].”
From Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition) (2021) · cites this paper as Lamoreaux et al. [174]
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References 27 · Cited by 268
- Authors
- Lamoreaux, L., Roederer, M., & Koup, R.
- Affiliations
- National Institutes of Health (US)
- Corresponding author
- Lamoreaux, Laurie
- Venue
- Nature Protocols
- Published
- 2006-08
- Volume
- 1
- Issue
- 3
- Pages
- 1507-1516
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- OpenAlex
- W2093264896
- Cited by
- 268
- References
- 27
- Citations by year
- 2026: 8 · 2025: 9 · 2024: 15 · 2023: 8 · 2022: 9 · 2021: 12 · 2020: 13 · 2019: 14 · 2018: 13 · 2017: 15 · 2016: 21 · 2015: 14 · 2014: 20 · 2013: 16 · 2012: 12
- FWCI
- 3.41
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
- Life Sciences › Immunology and Microbiology › Immunology › Immunotoxicology and immune responses
- Keywords
- intracellular cytokine staining, polychromatic flow cytometry, human T lymphocytes, compensation control, degranulation, T cell function
- MeSH terms
- Antigens, Surface, Flow Cytometry, Fluorescent Dyes, Humans, Immunologic Techniques, T-Lymphocytes, Cytokines
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