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J Immunol. 2015 Feb 15;194(4):2022-31. doi: 10.4049/jimmunol.1402661. Epub 2015 Jan 21.

Barcoding of live human peripheral blood mononuclear cells for multiplexed mass cytometry.

Author information

1
Human Immune Monitoring Center, Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA 94305; and hemei@stanford.edu.
2
Human Immune Monitoring Center, Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA 94305; and.
3
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.

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.

PMID:
25609839
PMCID:
PMC4323739
DOI:
10.4049/jimmunol.1402661
[Indexed for MEDLINE]
Free PMC Article

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