Mass cytometry: the user guide

History

Mass cytometry is a powerful technique initially developed by the University of Toronto and DVS Science (now Fluidigm) commercialised in 2009. Mass cytometry detects the presence of more than 40 specific extra or intracellular markers, enabling deep cell phenotyping. It is a version of flow cytometry, based on metals instead of fluorochromes. The resolution of this technique is so high that more than 40 metals (bound to antibodies detecting markers or antigens) can be detected at the same time. 

The first name of mass cytometry was CyTOF (Cytometry by Time of Flight), recently replaced by Helios while updating the CyTOF2 to CyTOF3.

 

The main steps to consider before performing mass cytometry analysis are: the staining, the acquisition and the data analysis.  

Antibody staining 

After dissociating tissue or processing blood,  the resulted single cell suspension is stained with antibodies, coupled to elemental isotopes. 

In nature, there are rare metal isotopes with a specific atomic mass. Specifically, lanthanides are chosen and the most used one is gadolinium. Naturally occurring gadolinium is composed of six stable isotopes, 154Gd, 155Gd, 156Gd, 157Gd, 158Gd and 160Gd, and one radioisotope, 152Gd not used in data analysis. There is indeed no radioactive elements to detect metals.  The metal isotopes used in mass cytometry are very pure to avoid background signals in channels.  

Each antibody is labeled with those pure metal tags, usually 5µg of metals is enough for 100µg of antibodies. Metals are first captured in specific polymers, which are then bound to the antibody by disulphide bridges. 

There are approximately 100 channels available to detect metals by mass cytometry. However, the number of purified metals does not exceed 55 at the moment, which means that the number of markers potentially screenable reached a maximum of 50, some metals being kept to detect viable cells, and others for barcoding for example. The metal conjugated antibodies are kept in a solution mix and then used to stain tissue. A DNA intercalator is added to the panel antibody mix to discriminate nucleated from non-nucleated cells. 

🚩 Tip: You can freeze your metal conjugated antibody mix and use it though your experiments. It will avoid some batch effects. 

 

 

Cell acquisition

After staining the cells usually overnight, the single cell suspension can be acquired at the CyTOF.  The acquisition is at least 20 times slower than conventional flow cytometers, but the number of parameters acquired is 3 to 4 times more important. You will gain depth in your data by using CyTOF. An efficient acquisition rate is around 300 events/sec. In this way, you can acquire a sample of 0,5 million cells in 30 minutes and prevent the formation of clogs in the sample lines. Most of the CyTOF-related-problems are the sample line clogging, especially just before cells enter the nebuliser or in the nebuliser line itself. 

During the acquisition, you can use some reference standard samples to check the staining quality. It is possible to acquire PBMC but you can also choose to stain and run VeriCells (BioLegend)

One of the optimum concentration to acquire the cells is 0,5 million cells/mL. Since the flow rate is 30µL/min, 900µL containing 0,5 million cells are processed into the CyTOF in 30 minutes. The single cells passed through the nebuliser, spraying them into the instrument itself. There, cells are burnt by an argon plasma, reaching the temperature of the sun, 7000°K. As a results, only a cloud of ions is left from the initial single cell solution. The light metals (< 75kDa) are removed and mainly constituted with biological elements. Only heavy metal ions are left, namely the lanthanides which are sorted based on their time-of-flight or TOF. Each ion signal is associated to a cell and every ion corresponds to a specific antibody recognising a marker. This results in a matrix where each individual cell is associated with an intensity level for every marker studied. 

Finally, FCS files are created for every sample acquired on CyTOF, comparable to flow cytometry. 

🚩 Tip: You can compensate your mass cytometry data to remove cross contamination background (optional). 

 

Online tools exist to facilitate creating your personalized conjugated antibody matrix. It is provided with an user guide. You need an account at Fluidigm to access it. 

Why should you use CyTOF in your research ?

 

1. A more intensive use in biomedicine

More and more labs are using mass cytometry to answer relevant biological questions. With mass cytometry, you can dive into the immune system mechanisms. A rapid expanding domain using mass cytometry is cancer.

For you, we summarised the publications on blood cancers, solid cancers, diabetes and we started to list COVID-19 studies as well. We mentioned the materials analysed, as well as the number of markers used.  

2. Deeper immune profiling: get more from your cells with better quality

CyTOF has revolutionised the way to investigate immune cells. Traditionally the number of maximum of parameters was set at 14 by using traditional flow cytometers. Now, the practical number of detectable markers reached 50, and theoretically can reach up to 100.  We summarise here the advantages of using CyTOF. 

No autofluorescence

When acquiring with CyTOF, you don't have to worry about autofluorescence coming from tumor cells or myeloid cells. If patients have been treated with some lanthanides, the corresponding metals should not be measured by CyTOF while processing the patient material. 

No compensation needed

You can measure your samples without compensating your data. There is an algorithm available to compensate CyTOF data if needed but this is not a requirement. All papers before 2018 were published without compensated data. The panel design is easier than for flow cytometry but some channels are more sensitive than others and the oxidation process is a source of cross-contamination between channels. You can check our page on mass cytometry analysis to know more. 

The highest number of parameters achieved

Mass cytometry presents the ability to resolve more than 100 metal probes. If in nature the number of purified metals which can be used now do not exceed 50, this number is continuously increasing. To date, even if technologies like CITE-seq are under development, it remains the most sensitive technology with the deepest immune profiling capabilities. 

3. Some alternatives to CyTOF does not reach equivalent data quality

New techniques like spectral flow cytometers are now being used and based on the full spectral detection. Approximately 30 markers can be investigated at the same time. Some flow cytometers claim performance with 40 different colors. If it might be the case to screen blood, it is more difficult with tumors, mainly because of intrinsic auto-fluorescence. Since CyTOF is based on lanthanides and that lanthanides are naturally not contained in tumors (except in case of specific treatments), CyTOF does not present any auto-fluorescence issues. 

One of the main advantages to use spectral flow cytometer is the data acquisition rate, which can be on average 15 000 cells/s, which is 50 times faster than CyTOF. 

We summarised here some pro and cons of CyTOF vs conventional flow cytometry vs spectral flow cytometry

Parameter
Conventional flow cytometry
Spectral flow cytometry
Mass cytometry
Number of parameters used
14
30
40
Maximum theoretical parameters
18
40
100
Optimal flow rate
14 000 events / sec
14 000 events / sec
300 events / sec
Time per sample
1 minute
1 minute
40 minutes
Comparison of the costs
+
++
++
Pre-work needed?
Set voltages, compensation
Run reference controls and calculate spectral unmixing
Compensation not needed
Preparation of samples
< 3 hours
< 3 hours
1 day (additional washing steps)
Autofluorescence
Try to be removed by compensation
Try to be removed by compensation
Not a problem
Spillover
Yes
Yes
Minimal
Clogging occurence
Not likely
Not likely
Very likely

Mass cytometry presents the great advantage to receive almost no background signal with an unprecedented number of parameters. We can help you analysing your data. Just send us your FCS files by filling our online form

The next step: towards a more integrated approach

On the genomic side, single-cell technology has improved the analysis of biological systems, via tumor genome sequencing (Navin et al., 2011; Vitak et al., 2017), tumor clonality dynamic (Yost, Satpathy et al. 2019), chromatin accessibility (Clark, Argelaguet et al. 2018) and even spatial positioning (Moffitt, Bambah-Mukku et al. 2018).  Single-cell RNA sequencing research was first used in a four-cell-stage blastomere (Tang, Barbacioru et al. 2009). At a larger scale, when used on e.g. the tumor microenvironment (TME), it allows for example a deep genotyping of every single cell present in the TME, allowing to genetically characterise different subsets (Azizi, Carr et al. 2018). 

A new way to investigate is to combine those different approaches together (Ha, Kwon et al. 2020). Some papers investigate the techniques cited above on the same tissue and assemble it for a comprehensive integration of single-cell data (Stuart, Butler et al. 2019).

A new technique currently proposed is sc-RNA-seq combined with mass cytometry or called CITE-seq. CITE-seq is an sc-RNA-seq technique with the addition of antibodies linked to DNA tag. This allows for immunophenotyping of cells with a potentially limitless number of markers and unbiased transcriptome analysis using existing single-cell sequencing approaches. However, this method is not sensitive and does not allow detection of low expressed molecules. TotalSeq from BioLegend are antibodies compatible with the CITE-seq technology. 

This trend shows that the quality and the depth of immune profiling is continuously increasing but CyTOF remains a very powerful immune profiling tool, with unprecedented detection capabilities conjugated with a large panel of detectable markers.