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Updated: Feb 19, 2026
Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry
Published on: June 26, 2018
Intracellular Cytokine Staining on PBMCs Using CyTOF™ Mass Cytometry
Dongxia Lin1, Sheena Gupta2, Holden T Maecker1,2
1Department of Microbiology and Immunology, Transplantation, and Infection, Stanford University, Stanford, USA.
Insights
This protocol details using mass cytometry for single-cell analysis of immune cells and cytokines. It optimizes intracellular cytokine staining for comprehensive immune profiling.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Mass cytometry (CyTOF™) enables high-dimensional single-cell analysis.
- Characterizing immune cell populations requires simultaneous detection of surface markers and intracellular cytokines.
- Existing protocols may have limitations in preserving cellular epitopes during permeabilization.
Purpose of the Study:
- To present a detailed protocol for intracellular cytokine staining using mass cytometry.
- To enable simultaneous analysis of a broad range of cytokines/chemokines and cell-surface proteins.
- To provide a compatible data output for various analysis software.
Main Methods:
- Cells are activated and treated with a protein transport inhibitor (e.g., Brefeldin A).
- Surface marker antibodies are applied, followed by fixation and permeabilization using saponin.
- Intracellular cytokine staining is performed using metal-conjugated antibodies before mass cytometry analysis.
Main Results:
- The protocol yields .txt and .fcs data files compatible with standard analysis programs.
- Saponin permeabilization preserves epitopes for robust antibody staining.
- The method allows for high-dimensional profiling of T cells and other immune cells.
Conclusions:
- This mass cytometry protocol provides a robust method for single-cell immune profiling.
- The protocol facilitates the simultaneous measurement of numerous cytokines and cell surface markers.
- The optimized staining and permeabilization steps enhance data quality for immune cell analysis.
Abstract:
In this protocol, we use a CyTOF™ mass cytometry to collect single-cell data on a large number of cytokines/chemokines as well as cell-surface proteins that characterize T cells and other immune cells. The current selected mass window in AW 103-203 includes the lanthanides used for most antibody labeling, along with iridium and rhodium for DNA intercalators. The output data are in the format as .txt and .fcs files, which is compatible with many analysis programs. This protocol could be adapted to include tetramers into the staining panel, but we have not optimized for that purpose. The principal steps of intracellular cytokine staining are as follows: First, cells are activated for a few hours using either a specific peptide or a non-specific activation cocktail. An inhibitor of protein transport (e.g. Brefeldin A) is added to retain the cytokines within the cell. Next, EDTA is added to remove adherent cells from the activation vessel. After washing, antibodies to cell surface markers are added to the cells. The cells are then fixed in paraformaldehyde and permeabilized. We use a gentle detergent, saponin, as the permealization buffer because it is less destructive to surface and intracellular epitopes compared to harsh detergents or methanol. After permeabilization, the metal-conjugated anti-cytokine antibodies are added into the cell suspension. The stained cells are then sequentially introduced into the mass cytometry for signal intensity analysis.
