Figure 1.

Multiplex analysis of antigen-specific T cells with mass cytometry based combinatorial tetramer staining. 1. Starting with a single blood or tissue sample, lymphocytes can be simultaneously stained with over 100 different combinatorially encoded heavy-metal labeled peptide-MHC tetramers. 2. To do this, tetramers are mixed into a single cocktail prior to cell staining. 3. The same cells are also stained with a cocktail of heavy-metal labeled antibodies for the purposes of probing the phenotypic and functional characteristics of the antigen-specific cells identified. 4. Prior to mass cytometry analysis, the cells are fixed, stained with metal-labled DNA interchelator, washed, and resuspended in water. 5. As they are introduced to the CyTOF^® mass cytometer, the cells are sprayed through a nebulizer and dried in argon before being ionized in plasma. Time-of-flight mass spectrometry is used to quantify each of the elemental tags on each cell. 6. Identities of antigen-specific cells are determined using multidimensional deconvolution algorithms and the phenotypic and functional characteristics of these cells can be compared through various analysis methods such as the clustergram shown.