title: Barcoding of live human peripheral blood mononuclear cells for multiplexed mass cytometry.
abstract: Mass cytometry is developing as a means of multiparametric single-cell analysis. In this study, we present an approach to barcoding separate live human PBMC samples for combined preparation and acquisition on a cytometry by time of flight instrument. Using six different anti-CD45 Ab conjugates labeled with Pd104, Pd106, Pd108, Pd110, In113, and In115, respectively, we barcoded up to 20 samples with unique combinations of exactly three different CD45 Ab tags. Cell events carrying more than or less than three different tags were excluded from analyses during Boolean data deconvolution, allowing for precise sample assignment and the electronic removal of cell aggregates. Data from barcoded samples matched data from corresponding individually stained and acquired samples, at cell event recoveries similar to individual sample analyses. The approach greatly reduced technical noise and minimizes unwanted cell doublet events in mass cytometry data, and it reduces wet work and Ab consumption. It also eliminates sample-to-sample carryover and the requirement of instrument cleaning between samples, thereby effectively reducing overall instrument runtime. Hence, CD45 barcoding facilitates accuracy of mass cytometric immunophenotyping studies, thus supporting biomarker discovery efforts, and it should be applicable to fluorescence flow cytometry as well.
authors.affiliation: Human Immune Monitoring Center, Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA 94305; and hemei@stanford.edu.
authors.forename: Henrik E
authors.initials: HE
authors.lastname: Mei
authors.affiliation: Human Immune Monitoring Center, Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA 94305; and.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Leipold
authors.affiliation: Human Immune Monitoring Center, Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA 94305; and.
authors.forename: Axel Ronald
authors.initials: AR
authors.lastname: Schulz
authors.affiliation: Human Immune Monitoring Center, Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA 94305; and Department of Oncology, Stanford University School of Medicine, Stanford, CA 94305.
authors.forename: Cariad
authors.initials: C
authors.lastname: Chester
authors.affiliation: Human Immune Monitoring Center, Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA 94305; and.
authors.forename: Holden T
authors.initials: HT
authors.lastname: Maecker
mesh_terms: D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D017493:Leukocyte Common Antigens; D007963:Leukocytes, Mononuclear; D013194:Staining and Labeling / Q000379:methods
chemical_list: D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human