The maximum number of simultaneous measurements being made on a single cell has been increasing rapidly in recent years. Fluorescence-based cytometers have achieved this with new and increased numbers of lasers and advances in fluorophore chemistry. Although the absolute limitations of fluorescence based cytometry panels keeps increasing, the practical limitation is far less for the vast majority of users. For example, reports of 27-color flow cytometry have been around for over a year, but the unavoidable fluorescence spillover and reduced resolution due to that spillover makes the practical limitation much less. The CyTOF-2 from Fluidigm Corp. has been able to leapfrog this limitation by using heavy metal isotopes to label antibodies rather than fluorescent tags and a time-of-flight mass spectrometer to detect and quantitate the metal tags. This affords the ability to combine many more antibody specificities in a single experiment, without significant spillover between detector channels. Together with metal-antibody conjugates that are created using chelating polymers and purified metal salts, or purchased as pre-made conjugates, the CyTOF allows for the creation of panels of up to 40 antibodies, along with live/dead markers, barcoding reagents, and a DNA intercalator to identify intact cells. Since the tags used on the CyTOF are not found naturally in anything typically run on a flow cytometer the background due to autofluorescence is essentially zero. This is a major advantage over flow cytometry especially in human samples that contain cells of the macrophage linage. Data from the CyTOF is exported as FCS files that can be analyzed using conventional flow cytometry software. However, the complexity of CyTOF data has led to the development of alternative analytical techniques, such as SPADE and ViSNE, which can be easily accessed and utilized with the commercial analysis software suite, Cytobank, Inc. Bringing this technology to the University of Chicago researchers will help expedite currently funded research projects that span many disciplines. Of special note is the utilization of the CyTOF-2 by the investigators in the Institut of Molecular Engineering (IME). One theme of the IME is focused on immune- bioengineering, which brings engineers, immunologists, and cancer researchers together for unique opportunities for advancement. Immunology is central to many of the questions of biological science and medicine and the University of Chicago has broad and deep strengths in it. As understanding of the immune system deepens, immune-bioengineering will become an increasingly powerful way to understand, manipulate, stimulate, and eventually control the immune system to address many conditions ranging from cancer and infections to allergies and autoimmune diseases.

Public Health Relevance

Thirteen NIH or privately funded projects focusing on biomedical and bioengineering research areas such as tumor immunity, autoimmune diseases, transplantation, pulmonary fibrosis, and vaccine development will rely heavily on the unique functionality the CyTOF-2 provides. State-of-the-art technologies employed on the requested instrument will help unveil the information necessary to answer these important Public Health questions.

National Institute of Health (NIH)
Office of The Director, National Institutes of Health (OD)
Biomedical Research Support Shared Instrumentation Grants (S10)
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Special Emphasis Panel (ZRG1)
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Levy, Abraham
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University of Chicago
Internal Medicine/Medicine
Schools of Medicine
United States
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