The CyTOF instrument is a mass spectrometer capable of high throughput sample injection.
The CyTOF instrument is similar to a traditional flow cytometer because it can screen numerous parameters on individual cells. However, it does so with the use of metal particle conjugated antibodies instead of the traditional antibody conjugated fluorophores that conventional flow cytometers uses.
Conventional flow cytometers, that use fluorescently conjugated antibodies to detect cellular antigens, have been practically maxed out at around 15-18 parameters. This is due to limitations in fluorophore technology. The spectral profile is limited in its capacity and there seems to be little room to force more fluorophores into the spectral profile at around 18 parameters. Newer fluorophores may eventually alleviate this issue.
Since the CyTOF machine detects heavy metals, in a process similar to mass spectrometry, the metals can be used as labels on antibodies instead of fluorophores. The number of metals available has been theoretically placed at around 100. Hence, many more parameters per cell can be measured at once when compared to fluorescent flow cytometry.
Therefore, the CyTOF has advantages over traditional fluorescent flow cytometry since it allows for a significant greater number of parameters to be studied per cell. The CyTOF is almost more sensitive and there is less error.
The disadvantages of the CyTOF is that cell are incinerated (so no sorting capabilities). Also, reagents are extremely expensive currently. Like any technology, price will likely come down as the technology is adopted.
- Greater number of parameters per event
- Higher sensitivity
- Little compensation required
- No “spreading” or error from fluorophores
- Higher cost to operate because of expensive reagents (and more per run)
- Limited conjugates available
- Can not sort
The CyTOF machine is solely distributed by DVS Sciences. DVS also sell reagents, but other companies such as eBio and BioLegend offer CyTOF specific reagents as well.
Figure shamelessly stolen from this article. I wasn’t a fan of the rest of the article, but the figure is nice!
Analyzing CyTOF Data
Since the majority of CyTOF users are acquiring greater than 20 parameters, most analysis is done with clustering algorithms. You can use traditional flow cytometry analysis techniques in FlowJo, but standard gating and population isolation becomes cumbersome when working with 20+ parameters. Therefore, most people are analyzing CyTOF data with clustering algorithms in Bioconductor. These are modules that are run through R. Some of the most popular clustering algorithms are SamSPECTRAL, flowMeans, and SPADE. One other package gaining ground recently is viSNE.
FlowJo can integrate with R and run SPADE analysis. This is a perfect integration because you can do your basic gating in FlowJo to clean up the data, modify compensation, adjust transformation, and then perform SPADE analysis. Integrating FlowJo with R is easy and is explained in this document:
Hooking up SPADE into R and running it through FlowJo is explained here:
There is a CyTOF user community available if you need more help. We posted the link recently on our daily dongle blog.
References and Resources