Overview

Mass cytometry 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.

Hyp 2 v3

The NIHR BRC Flow Core was the first to successfully establish a Mass Cytometry service in the UK and now operates two mass cytometry technology platforms, a Fluidigm HeliosTM  (see above right) for suspension cell work and a Fluidigm HyperionTM, imaging mass cytometer (see above left). Imaging mass cytometry uses the same basic principles of established suspension cell mass cytometry to analyze FFPE and frozen tissue sections.

The core offers a full service including custom antibody labeling, panel design, acquisition, troubleshooting and high dimensional data analysis, which is supported by a dedicated bioinformatician for custom analysis and a highly experienced team of mass cytometry specialists. In addition to standard analysis software the BRC Flow Core also has a licence to CytoBank (Spade, Visne, data sharing).

With over 100 detection channels, currently 38 stable isotopes for antibody tagging and more than10 markers for barcoding and cellular identity, we are able to deep phenotype clinical and research samples from internal and external clients. Staining protocols and workflows are similar to traditional fluorescent Flow Cytometry as shown in the figure below (modified from Fluidigm).

CYTOF diagram

The figure above gives a brief summary on how mass cytometry works. A liquid sample containing fixed cells labelled with metal-tagged antibodies/probes (A) is introduced into a nebulizer at a rate of ~500 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 analyzed using third party software as mentioned above (H).

Hyperion v2

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 analyzer (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 that 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 analyzed using third party software reconstructing the tissue architecture alongside high dimensional phenotyping (F).

Antibody conjugation service

The BRC flow core offers an antibody conjugation service for mass cytometry, bookable via ilabs The BRC Flow Core provides the metal conjugation kit and labelling consumables/reagents, manpower and expertise. The customer provides the antibody raw material (at least 100 ug of a protein free IgG formulation). We will conduct the conjugation and do a standard antibody QC (concentration and run of antibody on Helios for confirmation of successful metal tagging). Further biological testing will be done by the customer if and when required. External customers will discuss their project and provide a PO for the agreed project cost before the start of any work.

Further information

The BRC mass cytometers are operated as a facility service. All instruments and services are booked via iLabs. New users must register with iLabs (PDF 235Kb) and complete a Flow core registration form prior to first use. To reserve time on our mass cytometers, please make a booking on the instrument calendar via ilabs. The facility staff will review your request and confirm (or reject) your booking. You will receive a notification via ilabs Before the first mass cytometry experiment users are required to book a consultation session by contacting us via brcflowcore@gstt.nhs.uk.

To join the UK CyTOF user group please visit us at www.cytof.uk