Mass Cytometry
@ University of Manchester
@ University of Manchester
Mass cytometry, commonly referred to as CyTOF (Cytometry by Time of flight) is a single cell analytical technique that ustilises panels of antibodies conjugated to isotopically pure heavy metals. Detection is perfomred using a ICP-MS mass spec detection system and enables >50 parameters to be analysed in a single sample with minimal spill between channels. Additionally, up to 40 antibodies can be used to label tissue sections and cell cultures using the Hyperion Mass Imaging Cytometer.
Use the links at the top of the page to find out more information relating to this exciting new technique and look at the sections below for an introduction to what we can offer.
Before submitting samples to the facility to be run on the CyTOF please contact the facility manager to discuss your needs. We will discuss your sample prep, antibody panel and submission details.
We will also arrange for you to collect your antibody cocktail and reagents for labelling your cells that you may be using from the facility.
An appointment can be arranged with the CyTOF operator to run your samples.
Please submit an 'Order' for sample submission on the Facility's PPMS booking system at this link. When you create an order you will be required to complete a sample submission form.
Access to the facility is charged on an hourly rate in 15 minute blocks. The minimum charge is 30 minutes.
The CyTof samples at a fixed flow rate of 30ul per minute and up to a maximum of 500 events per second. Prior to a run cells are counted, adjusted to 1 x 10e6/mL and passed through a suitable filter to remove aggregated cells. We add EQ 'normalisation beads' to correct for instrument drift and then run the samples. As a guide it takes approximately 1 hour to run 1 million cells.
After samples have been collected we will perform the normalisation correction and send you the raw *.fsc files which you can upload to Cytobank for data analysis or run through any data analysis package you choose. See our data analysis page for extra information.
Designing a panel of antibodies is relatively straightforward for mass cytometry but there are a few considerations.
The detection of signal is optimal near the middle of the range of detection and this is where we would place antibodies to targets that are not highly expressed on cells or are up and down regulated upon stimulation.
A panel designing tool is available on the DVS Sciences page at www.dvssciences.com which allows you to construct a panel and submit it to Fluidigm for a quote.
Information on using the panel design tools can be found in this document Panel design presentation (Nina Lane, Fluidigm)
Data from the CyTOF is exported as flow cytometry standard (*.fcs) files and can be opened and analysed in third party programmes such as Flowjo.
However the multidimensional nature of the data often mean that users are better utilising clustering algorithms to look at their data. The facility has a cloud based solution to this in place for the initial MRC funding period of four years. We have subscribed to a solution called Cytobank.
Cytobank have posted two helpful blogs on how to use and what to consider when using these cluster analysis tools - How to Configure and Run a viSNE Analysis and Fine-Tune viSNE to Get the Most of Your Single-Cell Data Analysis
Cytobank tools
With Cytobank we have access to all of the current and future analysis solutions developed by the Nolan Lab in Stanford. These tools are based around cluster analysis algorithms that have been developed to identify low frequency cell events in multidimensional mass cytometry data.
SPADE, viSNE and heat maps
Cytofkit
An integrated mass cytometry data analysis pipeline that enables simultaneous illustration of cellular diversity and progression.
This runs within R and offers a user friendly interface. Additionally the output includes an PhenoGraph visualisation tool for analysing the data, as well as cluster maps.
Cytofkit can be downloaded from GitHub or Bioconductor
Chen et al (2016). “Cytofkit: A Bioconductor Package for an Integrated Mass Cytometry Data Analysis Pipeline.” PLOS Computational Biology, 12(9).
ACCENSE
A standalone tool for exploratory analysis of high-dimensional single-cell data such as that generated by Mass Cytometry and can be downloaded from the ACCENSE web page to run on Windows or Mac platforms
K Shekhar, et al (2014) Automatic classification of cellular expression by nonlinear stochastic embedding (ACCENSE) PNAS 111:202-7
We are running an antibody bank where users can place reagents they have purchased and gain 'credit' in the form of a number of tests. This credit then allow the researcher access to any antibodies currently available in the 'bank'.
Alternatively, the 'bank' allows new researchers access to this technology at a reduced cost. New users can buy a 'test' of an antibody from the bank and assemble a panel without having to make a large outlay on reagents.
Please contact the facility by email if you would like access to what reagents are available from the 'bank'