CyTOF Mass Cytometry for Click Proliferation Assays

Abstract

Novel cell analyzers, including polychromatic flow cytometers and isotopical cytometry by time of flight (CyTOF) mass cytometers, enable simultaneous measurement of virtually bondless characteristics at the single-cell level. BrdU assays for quantifying cellular proliferation are common but have several limitations, including the need for a DNA denaturation step and inability to simultaneously resolve multiple parameters and phenotypic complexity. Click chemistry reactions have become popular in the past decade, as they can resolve these issues. This protocol introduces a novel assay able to bridge flow cytometry and CyTOF analysis for active S-phase determination in cell cycle applications, combining well-established click chemistry with a novel iodo-deoxyuridine (IdU) azide derivative and a cross-reactive anti-IdU antibody for detecting incorporated EdU during DNA synthesis. This method is preferred over traditional BrdU-based assays for complex and multiparametric experiments. It provides a feasible cost-effective approach for detecting ethynyl-labeled nucleotides, with the advantage of combining flow and mass cytometry analyses. © 2017 by John Wiley & Sons, Inc.

Laser Capture Microdissection Coupled Capillary Immunoassay to Study the Expression of PCK-2 on Spatially-Resolved Islets of Rat Langerhans

“…In recent years, considerable efforts have been made to enhance the overall sensitivity of immunoassays for quantifying proteins. For example, multiple antibody-based methods for quantifying proteins in single cells have been developed such as CyTOF [20][21][22], scWestern blots [11,23], and Proseek Multiplex, an immunoassay readout by PCR [24]. Moreover, newly-developed ultrasensitive protein assays like immuno-PCR could be seen as an incremental step in the field of immunological research and clinical diagnostics.…”

Development of a Click-Chemistry Reagent Compatible with Mass Cytometry

“…Although another approach to allow detection of incorporated EdU with mass cytometry was recently published, we believe our approach to be more versatile 21 . This former approach did not use an azide-containing probe that can be loaded with any lanthanide ion and can be directly detected with mass cytometry, but instead used a 5-iodo-5′-azido-2′,5′-dideoxyuridine that contained an iodine molecule to perform click-chemistry 21 . In comparison, our approach is more generic and adaptable, and uses an azide-containing probe that can be loaded with any lanthanide ion of choice.…”

“…a lanthanide-tagged probe. Although a recent publication laid out one possible strategy to detect incorporated EdU with mass cytometry, we propose a more versatile strategy that allows labeling with a lanthanide ion of choice 21 .…”

Reagents for Mass Cytometry

“…Nucleoside mimetics continue to be a useful tool to monitor DNA synthesis in mass cytometry. 5-Ethynyl-2′-deoxyuridine (EdU, Figure ) and bromodeoxyuridine (BrdU) have been employed in similar strategies to IdU but with readouts via isotope-tagged Abs. , However, the use of BrdU is limited due to the Ar dimer ( m / z 80) potentially interfering with Br isotopes ( m / z 79 and 81). As such, IdU is used to avoid such interference.…”

“…Metabolic substrates used for monitoring dynamic cellular states in mass cytometry. Thymidine analogs, (A) EdU (5-ethynyl-2′-deoxyuridine) and (B) IdU (5-iodo-2′-deoxyuridine), are both used to identify S-phase cells in mixed populations. Cellular DNA replication machinery incorporates EdU and IdU during S-phase; while EdU requires a lanthanide-tagged azide for click-chemistry mediated detection, IdU incorporation can be measured directly with the m / z 127 channel.…”