Mass Cytometry is a relatively new technology which has recently featured in many high-impact journals. You may have read about instruments including the CyTOF, CyTOF2, and more recently, the Helios.
With these instruments becoming more widespread, you might find yourself asking, what is mass cytometry, and what can it do for you?
The Basis: Conventional Flow Cytometry
In conventional flow cytometry, antibodies specific for your protein of interest are conjugated to fluorescent molecules. When cells labeled with these antibodies pass through a flow cytometer, lasers excite the fluorescent molecules and they produce a signal corresponding to the markers expressed on each cell, generating a specific cellular profile.
Flow cytometry is an invaluable technique across many scientific fields, but is limited to investigating up to 16 fluorescent parameters per cell, depending on the sophistication of your available flow cytometer. The major limiting factor in flow cytometry is that of spectral overlap between different fluorescent labels and the loss of data quality when compensating for this overlap.
Bring on the Mass
Mass cytometry overcomes these limitations by conjugating antibodies to stable isotopes of heavy metals, instead of fluorescent molecules. Mass cytometers use the “Time-Of-Flight” (TOF) mass spectrometry method, in which these heavy metal probes give signal in only one mass channel. This eliminates the problem of spectral overlap so you can analyze more than 50 parameters in a single sample. Think of all the information you can obtain from just one tube!
Fortunately, with mass cytometry you don’t need to worry about analyzing complex mass spectrometry data, as the software kindly produces an FCS (flow cytometry standard) file for you to take away and analyze using your favorite flow analysis software.
However, you should also explore exciting bioinformatics tools like viSNE and SPADE tree analysis, which will enable you to visualize your data, extract statistical information, and identify rare cell populations much more effectively. These analyses can be carried out using Cytobank software or, for those of you are a little more computer savvy, packages using the R programming language.
Working with Metals
Right now, you can only buy metal-conjugated antibodies from Fluidigm, which has a number of pre-designed panels available as well as a growing catalog of individual antibodies. If they don’t have the marker you need, it’s quite straightforward to conjugate your own antibody to the metal of your choice. A number of companies now have a range of “CyTOF-ready” purified antibodies for this purpose. You can also ask your Fluidigm representative if your antibody of interest is in the pipeline, as new reagents are being developed all the time. Cell surface marker antibodies and antibodies for intracellular staining, for measuring cytokines or phosphorylation, are available. Reagents are also available for cell cycle analysis, and you can also include that all-important viability dye in your assay.
Another great tool that can be applied in mass cytometry is barcoding. You can combine a number of samples marked with different “barcodes” so you can mix them as one sample and stimulate (e.g., with cytokine), stain, and acquire them together. This makes sample preparation much quicker and easier and reduces variability between samples. After acquisition, a software algorithm will deconvolute the data ready for you to carry out analysis of the individual samples.
The development of new reagents and protocols is making mass cytometry more versatile than ever, revealing its potential as a unique and truly powerful technique. Why don’t you contact your nearest operator and see what it could do for your projects?