Validation of Click Chemistry with Mass Cytometry for Single Cell Analysis

Abstract

The field of molecular biology has rapidly advanced since the development of single cell characterization technologies, such as flow cytometry and monoclonal antibodies. More recently, novel single cell characterization methods have been developed to address the limitations of previous technologies. Mass cytometry detects the presence of antigen based on the molecular mass of a probe, sidestepping the problem of overlapping fluorescent signals in flow cytometry. Currently, mainly antibody‐based assays are possible for analysis on a mass cytometer. Here, we developed a novel probe that allows identification and detection of events using click chemistry. Click chemistry involves a method of labeling reagents in a stable, covalent way that circumvents the need for cumbersome antibody conjugations to metal probes and fastidious tittering and validation. Here we show that the thymidine analog 5‐ethynyl‐2′‐deoxyuridine (EdU) can be accurately detected via mass cytometry when labeled with a metal‐tagged azide in a click reaction. The level of detection is as expected based on comparative experiments analyzed using flow cytometry, showing that the combined use of click chemistry and mass cytometry is both feasible and valid. Furthermore, it paves the way for future experiments combining these techniques. Mass cytometry can theoretically measure up to about 100 parameters at once, many more than possible with flow cytometry, and without the need for laborious compensation. This feature makes it particularly attractive to immunologists and pharmacologists who are studying the intricate subtypes of immune cells and signaling cascades involving many parameters. In addition, click chemistry provides a very simple, easy staining procedure that is highly specific and compatible with many markers such as antibodies but could be especially valuable to detect non‐protein events. We believe the addition of click‐chemistry to the mass cytometry toolbox allows for expanded utility of these methods and will prove invaluable as molecular biologists probe deeper into our physiology.