Customizing Maxpar Direct Immune Profiling Assay with Additional Surface Marker and Intracellular Cytokine Staining Workflows for Expanded Mass Cytometry Panels.

Mass cytometry, or cytometry by time-of-flight (the basis for Fluidigm® CyTOF® technology), is a system for single-cell detection using antibodies tagged with metal probes. Without the need for compensation, the highly parametric Helios™ mass cytometer has a detection range of 135 distinct mass channels (75-209 Da). Optimized for mass cytometry, the Maxpar® Direct™ Immune Profiling Assay™ is a dry, metal-tagged antibody cocktail for immunophenotyping 37 immune cell populations found in human peripheral blood in a single tube. The Maxpar Direct Assay utilizes 31 mass channels for marker detection and live/dead viability staining, with at least 14 additional marker channels available from the Fluidigm catalog for flexible custom panel design. Here, we describe a workflow combining the assay with additional surface and intracellular cytokine antibodies for peripheral blood mononuclear cell (PBMC) staining using lanthanide-, bismuth-, and cadmium-tagged antibodies.

Mass Cytometry for the Characterization of Individual Cell Types in Ovarian Solid Tumors.

Mass cytometry aka Cytometry by Time-Of-Flight (CyTOF) is one of several recently developed multiparametric single-cell technologies designed to address cellular heterogeneity within healthy and diseased tissue. Mass cytometry is an adaptation of flow cytometry in which antibodies are labeled with stable heavy metal isotopes and the readout is by time-of-flight mass spectrometry. With minimal spillover between channels, mass cytometry enables readouts of up to 60 parameters per single cell. Critically, mass cytometry can identify minority cell populations that are lost in bulk tissue analysis. Mass cytometry has been used to great effect for the study of immune cells. We have extended its use to examine single cells within disaggregated solid tissues, specifically freshly resected tubo-ovarian high-grade serous tumors. Here we detail our protocols designed to ensure the production of high-quality single-cell datasets. The methodology can be modified to accommodate the study of other solid tissues.

Method for Tagging Antibodies with Metals for Mass Cytometry Experiments.

Mass cytometers are time-of-flight (TOF) mass spectrometer-coupled flow cytometers (known as CyTOFs) that quantify the abundance of metal-tagged antibodies (Abs) or other cellular probes within single cell suspensions or laser-ablated tissue sections. While many strategies exist for covalently crosslinking to proteins, the Fluidigm MaxPar® process is currently the most widely used and involves first loading a metal-chelating polymer with an elementally and isotopically enriched metal. Once the chelation sites have been filled, a maleimide moiety on the polymer is reacted with the free thiol groups on the partially reduced monoclonal immunoglobulin G (IgG) Ab to form an irreversible covalent bond. Here we describe modifications to the Fluidigm MaxPar® protocol that increase the quantity of Ab per reaction up to 150 µg, introduce an initial Ab quality control step, utilize metal labels not included in the Fluidigm catalog, and provide an option to perform two reactions in one centrifugal filter.

Immunophenotyping of Human Peripheral Blood Mononuclear Cells by Mass Cytometry.

Mass cytometry is a variation of conventional flow cytometry using metal tagged antibodies for cell staining instead of fluorochromes and detection in a mass cytometer, a modified mass spectrometer that allows for separation of discrete masses of these metal tags by time of flight (TOF). Currently, up to 50 different metal tags are available for cell analysis. The lack of any significant mass spectral overlap and autofluorescence background makes mass cytometry uniquely suited for complex high-dimensional phenotypic and functional analysis at the single cell level, thus accelerating biomarker discovery and drug screening. Here we describe a workflow for phenotyping of human peripheral blood mononuclear cells (PBMCs) covering cell staining, instrument setup of a Fluidigm Helios™ mass cytometer, and sample acquisition, and summarize a basic workflow of data analysis.