The CyTOF Flow Core will deploy the recently introduced mass spectrometry - flow cytometry hybrid instrument for the use by participants in the grant. CyTOF (CYtology Time Of Flight) is a newly developed single cell analysis instrument which replaces fluorophores with mass tags. The utility of this approach is that there is an immediate 10 fold increase in the number of parameters that one can measure during nearly any experimental flow cytometry procedure. Purified isotopes in the lanthanide series are attached to antibodies by way of a specific chelator and then used to stain cells in a manner similar to traditional fluorescence based flow cytometry. Not only is there a great increase in the number of parameters that can be measured I per cell basis, but critically no fluorescence compensation is required nor is there any autofluorescence. This greatly increases the accuracy and statistical value of the information gathered on a per cell basis. The flow core will bring this technology to the project as a whole in the analysis of murine immune system mutations generated by the experimental projects to determine on a whole immune system basis both the subtle and gross changes to immune architecture governed by mutant genes discovered in the experimental projects, or caused by pathogens tested on such mutant mice. We have over 300 verified antibodies (surface markers, intracellualar phosphorylation state markers, etc.) that have been verified to work by this procedure, robotic staining systems, advanced analytic tools which allow for ready mining and interaction with the data, and an in-house developed online web-based flow cytometry analysis suite that is also fully capable of password protected advanced, HIPPA compliant secure storage. Materials sent to us, or animals, can be analyzed at a depth that no traditional flow cytometry facility can accomplish, including not only multiple surface markers but as well as many signaling markers intracellularly as we require. Another recent advance in our group has been a demonstration of single cell flow cytometry measurement of RNA down to as few as five molecules per cell and with a dynamic range of 100,000 fold. In the years of this grant we will bring this and other outlined capabilities to the experimental projects to enable the discovery process.

Public Health Relevance

Cytometry Time of Flight (CyTOF) is a proteomic technology measuring cellular function in specific cell subsets of the immune system. Traditional flow cytometry measures about 8 parameters per cell;CyTOF measures >35 parameters per cell, providing the detail necessary to study the murine immune system in the context of the mutations in this proposal. This information can then be applied to the human immune system.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Program--Cooperative Agreements (U19)
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Scripps Research Institute
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