Immunology

Single-cell analysis has become an method of importance in immunology. Fluorescence flow cytometry has been a major player. However, due to issues such as autofluorescence and emission spillover between different fluorophores, alternative techniques are being developed. In recent years, mass cytometry has emerged, wherein antibodies labeled with metal ions are detected by ICP-MS. In order for a cell to be seen, a metal in the mass window must be present; there is no analogous parameter to forward or side scatter. The current mass window selected is approximately AW 103-196, which includes the lanthanides used for most antibody labeling, as well as iridium and rhodium for DNA intercalators.

In this protocol, we use a cocktail of antibodies labeled with MAXPAR metal-chelating polymers to surface-stain live PBMC that have been previously cryopreserved. Many of these markers were taken from a standard fluorescence phenotyping panel (Maecker et al., 2012). No intracellular antibodies are used. We use a CyTOF^TM (Cytometry by Time-Of-Flight) mass cytometer to acquire the ICP-MS data. Subsequent analysis of the dual count signal data using FlowJo software allows for cell types to be analyzed based on the dual count signal in each mass channel. The percentage of each cell type is determined and reported as a percent of the parent cell type.

In this protocol, we use a CyTOF^TM 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.