Mass cytometry

Mass cytometry is made available at the Charles Perkins Centre through a collaborative effort between the Sydney Cytometry Facility and the Ramaciotti Facility for Human Systems Biology (RFHSB, https://www.ramaciotti-hsb.org.au). The RFHSB was established through the 2013 Clive and Vera Ramaciotti Biomedical Research Award with the goal of developing systems level understanding of human disease via integration of highly multiparametric mass cytometric data with clinical and other data sources. It facilitates user access to mass cytometry and imaging platforms via training and reagent development, and supports data analysis pipelines.

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What is mass cytometry?

Put simply, mass cytometry replaces the fluorescent reporters used in fluorescence flow cytometry, with metal isotopes, which are detected using cytometry by time-of-flight mass spectrometry (CyTOF). This has expanded the number of parameters that can be detected simultaneously on single cells to approximately 40 (with >100 theoretically achievable), due to the absence of signal overlap between metal isotopes. This allows an investigator to measure an enormous number of extracellular and intracellular targets simultaneously.

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How it works

The 'mass cytometry' methodology relies on the distinct atomic masses of individual metal isotypes, which are conjugated to antibodies specific for surface or intracellular antigens on single cells. Labelled cells are introduced into the machine by generating a liquid aerosol through a nebulizer, which passes sample through an argon-fuelled inductively-coupled plasma (ICP) torch, burning at approximately 7500 K. The ensuing ion cloud is then drawn into a vacuum, where ions of less than atomic mass 80 are removed from the samples, before the samples enter the time-of-flight chamber. This form of mass spectrometry ensures that metal ions with different atomic masses will arrive at the detector at different times, separated by the flight time required to pass through the chamber. Ions with larger atomic mass take longer to arrive than those of lower atomic mass. The resulting data is high-dimensional single-cell data, consisting of clean and distinct metal abundance peaks that correlate with cellular antigen expression.

For more information on mass cytometry at the Ramaciotti Facility for Human Systems Biology (RFHSB), and the Sydney Cytometry facility, see this ASI newsletter.

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Types of mass cytometry

Two formats of mass cytometry are available at Sydney Cytometry: suspension (HELIOS) and imaging (HYPERION, IMC). Suspension mass cytometry involves staining samples in solution with a cocktail of metal-conjugated antibodies, and acquiring them in a solution format (similar to flow cytometry). Imaging mass cytometry involves labelling frozen or formalin-fixed paraffin-embedded (FFPE) tissue sections with metal-conjugated antibodies, and using laser ablation to release the metals tags from the slide for analysis.

 

Getting started with mass cytometry

 
 

INITIAL MEETING

To start a mass cytometry project (suspension or imaging mass cytometry), you will first need to meet with the operations team to work out the details of your experiment. You can arrange a meeting by submitting a new project request.

 

PPMS REGISTRATION, BIOSAFETY APPROVAL, AND CYTOF INTRODUCTORY TRAINING

After meeting with members of the operations team, you will need to register with PPMS, submit a biosafety approval request, and request CyTOF training. All of these steps, with the relevant links, are outlined here

The first level of CyTOF training allows users to run samples on the cytometer under supervision, during work hours. Once a sufficient level of competency has been reach, users can operate the cytometer unsupervised. Alternatively, facility staff may be engaged to acquire samples on behalf of the user at cost (rates available here, under 'Operator Assistance'.