Part 2. CyTOF Assays
Most of the same techniques and reporter molecules can be used in CyTOF assays as in fluorescence flow cytometry. While antibody conjugates are the most common reagents, other reporters such as live/dead or cell cycle reagents are also possible to use. In our lab, we run three major assay types: immunophenotyping, intracellular cytokine staining, and phospho-signaling. These assays differ in the use and timing of in vitro stimulation, such that they can be considered as independent experiment types that usually cannot be combined.
- This assay is done without in vitro stimulation, and can be used for enumeration of many different phenotypes of immune cells. While our protocol is based only on cell-surface markers, it is possible to add intracellular markers that do not require stimulation. These could include cytotoxic granule proteins like perforin or granzyme, or transcription factors like FoxP3, Tbet, GATA3, or RORgt. However, these latter require harsh permeabilization that can degrade the surface staining antibodies already applied. Hence, we prefer to use chemokine receptors to give a rough indication of T helper profiles (though chemokine receptors are themselves not highly expressed, and may not correlate very precisely with Th cytokine profiles).
- When comparing frequencies of cell types between samples or groups, it is helpful to express them as frequency of a higher-order parent population, such as live intact single cells. In this way, the effects of changes in frequencies of other cell types with the same parent population will have less impact on the data. For example, expressing CD4+ T cells as a frequency of total T cells will be greatly influenced by changes in frequency of CD8+ T cells, but this will be less noticeable if expressing as a frequency of all live intact cells.
- A basic protocol for CyTOF phenotyping of PBMC. (Leipold and al., Bio-protocol, 2015)
- A more extensive book chapter with some review of panel design and issues to watch out for. (Leipold and al., Immunosenescence, 2015)
Intracellular cytokine staining (ICS):
- Readout of intracellular cytokines is generally done with 4 h or longer in vitro stimulation, in the presence of brefeldin A and/or monensin to inhibit cytokine secretion. Stimulation can be with specific antigens (peptides or proteins), or with non-specific stimuli such as LPS or other TLR ligands, CD3+CD28, or PMA+ionomycin. The latter is useful for reading out the full potential of cytokines and activation markers from essentially all immune cell types: T cells, B cells, NK cells, monocytes, and dendritic cells. We refer to this as measuring “global immune competence”. 4 h stimulation with PMA+ionomycin is usually ideal, to avoid excessive cell death, while stimulation with specific antigens is usually longer, often 6-8 h or even overnight.
- For readout of CD154 (CD40L) and CD107a (marker of degranulation), we typically add the staining antibodies for these markers to the stimulation culture. This allows for staining even if the target molecules are rapidly endocytosed after expression on the cell surface. Monensin (5 ug/ml) should be used to prevent acidification of endocytic vesicles, which could break down the staining antibodies. This can be used in addition to 5 ug/ml brefeldin A, which is the preferred secretion inhibitor for cytokines like IFNg, TNFa, and IL-2.
- Cell lineage markers and other cell-surface phenotyping markers are stained prior to permeabilization and staining for cytokines and other intracellular markers, so this assay provides both phenotypic and functional readouts. We recently developed a version of the assay that can be performed on fixed whole blood, allowing the collection and/or stimulation of samples to be disconnected from the staining and acquisition.
- A basic protocol for CyTOF ICS. (PBMC) (Lin, Methods in Molecular Biology, 2015)
- A basic protocol for CyTOF ICS (Fixed Whole Blood) and animation below. (Kowli et al., Bioprotocol, 2020)
- Mass Cytometry Assays for Antigen-Specific T Cells Using CyTOF (Lin D and Maecker HT, 2018, Methods Mol. Biol.)
- Application of Mass Cytometry (CyTOF) for Functional and Phenotypic Analysis of Natural Killer Cells (Kay AW. et. al., 2016, Methods Mol. Biol.)
- A more extensive book chapter showing how CyTOF ICS can be used for comprehensive immune competence testing. (Subrahmanyam, Current Protocol Cytometry, 2018)
This assay reads out the phosphorylation state of multiple phosphoprotein epitopes, together with cell-surface phenotyping for identification and enumeration of major cell lineages. Stimulation times are usually short, e.g., 15 min, using cytokines, PMA+ionomycin, TLR ligands, etc. The phospho-epitope readouts are usually quantified as the difference in intensity between stimulated and unstimulated conditions, using an optimized arcsinh transformed scale (see http://support.cytobank.org/help/kb/building-illustrations/what-is-the-arcsinh-transform). Cytobank produces heat maps and histogram overlays that are particularly well-suited to displaying phospho-CyTOF data. Meanwhile, the assay also enumerates cell phenotypes, which are generally quantified as frequencies.
Because of the need to preserve the phospho-signaling state of the cells at the end of the stimulation, cells are generally fixed prior to cell-surface staining. Thus, this assay requires the use of antibodies to fixation-resistant epitopes. While generally not an issue for most cell lineage markers, this requirement can make it difficult to visualize chemokine receptors and other markers that are poorly expressed and/or for which antibodies only recognize native epitopes.
Protocols for phospho-CyTOF of PBMC and whole blood have been published:
For PBMC. (Fernandez, Bio Protocol, 2016)
For whole blood. (Fernandez, Bio Protocol, 2016)