Dual‐labelled antibodies for flow and mass cytometry: A new tool for cross‐platform comparison and enrichment of target cells for mass cytometry. Issue 8 (18th July 2017)

• Record Type: Journal Article
• Authors: Baumgart, Sabine; Schulz, Axel R; Peddinghaus, Anette; Stanislawiak, Silke; Gillert, Sarah; Hirseland, Heike; Krauthäuser, Susanne; Dose, Christian; Mei, Henrik E; Grützkau, Andreas
• Is Part Of: European journal of immunology. Volume 47:Issue 8(2017)
• Page Start: 1377
• Page End: 1385
• Publication Date: 2017-07-18
• Subjects: Cell enrichment -- Cross‐platform -- CyTOF -- Dual conjugates -- Dual‐labelled antibodies -- FACS -- Flow cytometry -- Mass cytometry -- Plasmablasts -- Rare cell population -- Standardisation; Immunology -- Periodicals; 616.079
• DOI: 10.1002/eji.201747031
• Languages: English
• ISSNs: 0014-2980

Abstract

Abstract : Here, the preparation and application of dual‐labelled antibodies carrying a fluorochrome molecule and metal‐chelated polymers are described, which are useful tools for the enrichment of rare cell populations by FACS at first before deep phenotyping follows by mass cytometric analysis (CyTOF). Abstract : Antibody conjugates applicable in both conventional flow and mass cytometry would offer interesting options for cross‐platform comparison, as well as the enrichment of rare target cells by conventional flow cytometry (FC) sorting prior to deep phenotyping by mass cytometry (MC). Here, we introduce a simple method to generate dual fluorochrome/metal‐labelled antibodies by consecutive orthogonal labelling. First, we compared different fluorochrome‐conjugated antibodies specific for CD4, such as FITC, Vio667, VioGreen or VioBlue for their compatibility with the conventional secondary MAXPAR ® labelling protocol. After labelling with 141 Pr, the fluorescence emission spectra of all fluorochromes investigated retained their characteristics, and CD4 dual conjugates (DCs) provided consistent results in immune phenotyping assays performed by FC and MC. The phenotypical composition of CD4 + T‐cells was maintained after enrichment by FC sorting using different CD4 DCs. Finally, magnetic cell depletion was combined with FC sorting using CD19‐VioBlue‐ 142 Nd, CD20‐VioGreen‐ 147 Sm, CD27‐Cy5‐ 167 Er and CD38‐Alexa488‐ 143 Nd DC to enrich rare human plasmablasts to purities >80%, which allowed a subsequent deep phenotyping by MC. In conclusion, DCs have been successfully established for direct assay comparison between FC and MC, and help to minimise MC data acquisition time for deep phenotyping of rare cell subsets.