Mass cytometry (CyTOF) is a variation of conventional flow cytometry using metal tagged antibodies instead of fluorochromes and detection by time of flight of discrete masses of the metal tags.
The lack of any significant mass spectral overlap (no need for compensation) and auto-fluorescence background makes this technology uniquely suited for unravelling high dimensional functional and phenotypic correlations at the single cell level, accelerating biomarker discovery and drug screening.
Our Flow Core was the first to successfully establish a Mass Cytometry service in the UK and now operates two mass cytometry technologies, a Standard BioTools (formerly Fluidigm) "Helios" for suspension cell work and a "Hyperion" imaging mass cytometer.
Imaging mass cytometry uses the same basic principles of established suspension cell mass cytometry to analyze FFPE and frozen tissue sections.
The core offers services for both modes, including custom antibody labelling, panel design, acquisition, troubleshooting and high dimensional data analysis.
With currently 35 stable lanthanide isotopes, as well as Cadmium, Yttrium and Bismuth isotopes for antibody tagging, in addition to more than 10 markers for barcoding and cellular identity, we are able to perform deep phenotyping on clinical and research samples from internal and external clients. Staining protocols and workflows are similar to traditional fluorescent Flow Cytometry.
Our instruments:
Helios A is configured solely in liquid mode (for single cell analysis), similar to conventional flow cytometry but with metal-labelled reagents.
Helios B is normally configured with the optional Hyperion unit for Imaging Mass Cytometry (i.e. slides), similar to conventional microscopy on FFPE or fresh-frozen tissue sections/ microarrays, but with metal-labelled reagents. This instrument can be run in standard Helios mode as and when required.
The figure above gives a brief summary on how liquid mass cytometry works. A single cell suspension of fixed cells labelled with metal-tagged antibodies/probes (A) is introduced into a nebulizer at a rate of ~300 cells/sec creating an aerosol (B) and then transported towards an argon plasma (C) where the cells are vaporized, atomized and ionized. After selective removal of low mass ions in the quadrupole (D), the ion cloud containing the isotopic metal tags used for labelling enters the TOF chamber where probes are separated by time of flight based on their mass to charge ratio as they accelerate towards the detector (E). The time-resolved detector measures a mass spectrum that corresponding to the identity and quantity of each isotopic probe on a per-cell basis (F). Data is saved in FCS format (G) and can be analysed using third party software (H).
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The figure above gives a brief summary on how imaging mass cytometry works. A tissue sample containing fixed tissue/cells labelled with metal-tagged antibodies/probes (A) is introduced via autostage into the Hyperion Imaging analyser (B) and ablated with a high power pulsed UV laser focused on a 1mm spot size and operating at 100-200 Hz, resulting in plumes of ablated tissue atoms in an inert carrier gas (C) which are then transported towards an argon plasma of the Helios mass cytometer (D) where the arriving atoms are ionized. After selective removal of low mass ions in the quadrupole (D), the ion cloud containing the isotopic metal tags used for labelling enters the TOF chamber where probes are separated by time of flight based on their mass to charge ratio as they accelerate towards the detector (E). The time-resolved detector measures a mass spectrum corresponding to the identity and quantity of each isotopic probe on a per-cell basis (E). Data is saved in MCD and TIFF format and can be analysed using third party software reconstructing the tissue architecture alongside high dimensional phenotyping (F).
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The Flow Core offer an antibody conjugation service for mass cytometry, self-bookable via iLab (please contact us in advance to discuss your needs and our availability). The facility provides the conjugation kit for tagging with lanthanide metals (mass range 141-176), all labeling consumables/reagents, labour and expertise. The customer is requested to provide the antibody raw material. We require at least 100 mg of a protein free IgG solution, the facility will not purify or conjugate antibodies provided in BSA/ gelatin containing buffer. We will return the metal conjugated antibody in protein stabilizing solution alongside a quality control data sheet with proof of concentration and successful metal conjugation (biological testing is to be done at customer site). External customers will discuss their project and provide a PO for the agreed project cost before the start of any work.
All mass cytometry platforms and services are operated by fully trained staff only.
Instruments and services are booked via iLab. New users must register with the platform then sign up with iLab prior to first use. To reserve time on our mass cytometers, please make a booking on the instrument calendar. The facility staff will review your request and confirm (or reject) your booking, you will receive a notification via iLab. Before the first mass cytometry experiment users are required to book a consultation session by contacting us via BRCFlowCore@gstt.nhs.uk.