Writing a CyTOF 2 Grant Application

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1 PN A2 TECHNICAL NOTE Writing a CyTOF 2 Grant Application This document provides general guidelines for writing a CyTOF 2 instrument grant application. The guidelines are broken into sections that correspond to the structure of an NIH Shared Instrumentation Grant. Because mass cytometry is breakthrough technology, place special emphasis in two areas. First, demonstrate a clear understanding of the unique, research-advancing capabilities the platform provides. Second, outline a rational infrastructure and plan for successfully integrating the platform into the research goals of the institution post sale. It is generally expected that all proposed research projects include a high-dimensional panel. In addition, include one small pilot dataset (2 samples max) collected on a CyTOF or CyTOF 2 instrument to demonstrate understanding of mass cytometry workflow. Therefore it is important to start the process early, at least six months prior to the deadline. As with any grant process, it is important to not only start early but also follow the grant instructions carefully, and use your own words. Justification of Need Here it is important to articulate the unique technological attributes of the CyTOF 2 platform and how they translate into beneficial research-advancing experiments that cannot be done with existing technologies. Convey that your users currently do not have access to the technology at or near your institution. It is also useful to indicate how you will employ the mass cytometer along with other techniques (such as flow cytometry, magnetic enrichment, quantitative PCR, etc.) to which you already have access to achieve the aims in the proposal. Below is some example text: Cells express a complex array of components that indicate physiologic status, phenotype and functional capability. Therefore, platforms that provide multiparametric cellular analysis are uniquely suited to. CyTOF is uniquely capable of simultaneously resolving more than 35 probes on a per-cell basis at high acquisition rates, thereby providing researchers with an unparalleled ability to phenotypically and functionally profile cells from normal and diseased states. Instead of fluorescence-based cytometry, mass cytometry employs element-tagged probes that are measured on the CyTOF 2 instrument, which resolves isotopes of different masses with minimal signal overlap over an extended mass range. Furthermore, mass cytometry uses rare earth elemental tags which provide similar signal intensities to one another, and are not naturally found in cells, thereby avoiding measurement of background cellular signal. All of these attributes simplify large panel

2 experimental design, thereby uniquely enabling high-dimensional cytometry experiments not possible with other methods. Acquisition of the CyTOF 2 Mass Cytometry platform will uniquely position our researchers to advance the understanding of.conditions pertaining to our studies include The following reviews provide examples of relevant discussions of the unique benefits of this technology: Feinberg, H.G., Nolan G.P. Mass cytometry to decipher the mechanism of nongenetic drug resistance in cancer. Current Topics in Microbiology and Immunology 377 (2014) Bendall S. and Nolan, G.P. From single cells to deep phenotypes in cancer. Nature Biotechnology 30(7) (2012): 639. Benoist C.. Flow Cytometry, Amped Up. Science 332 (2011): 677. Doerr A. A flow cytometry revolution. Nature Methods 8(7)(2011): 531. Janes M.R. and Rommel C. Next-generation flow cytometry. Nature Biotechnology 29(7) (2011): 602. Nolan G. Flow cytometry in the post fluorescence era. Best Practice & Research Clinical Haematology 24 (2011) A more complete listing of publications is available at 2 Writing a CyTOF 2 Grant Application

3 Justification of Need for Fluidigm Cytobank The high-dimensional data sets generated by CyTOF 2 are best analyzed with software that provides easy-to-use tools to interpret and present multi-parametric analysis. Fluidigm Cytobank is our recommended data management and analysis solution. If you choose to use Fluidigm Cytobank, either include it in the cost of the grant proposal and add justification or demonstrate institutional funds/support for acquisition and maintenance of this software. An overview of Fluidigm Cytobank is available at Fluidigm Cytobank ( enables users to manage and analyze the large amounts of mass cytometry data over the web. It has tools designed to handle the three layers of analysis common to high-dimensional mass cytometry experiments. These include: raw data plotting tools such as histograms (and histogram overlays), dot plots, and gating; dimensionality reduction algorithms critical to rendering multidimensional data onto two-dimensional space (SPADE and visne); and statistical representations of multiple samples such as heatmaps and dose response curves. Furthermore, Fluidigm Cytobank provides a centralized, secured, annotated database of all recorded mass cytometry data and facilitates internal and multi-site collaborations. Fluidigm Cytobank meets the data sharing requirements outlined in section 8.2 of the NIH Grants Policy on Availability of Research Results: Publications, Intellectual Property Rights, and Sharing Biomedical Research Resources ( by providing sharing containers to make published data and results available to the community. Research Projects Here it is important to have a set of projects that clearly link the unique benefits of mass cytometry to the proposed research, and that are planned in sufficient detail to indicate basic understanding of the practical considerations required for using the system in the research. The projects should not be achievable with existing or alternatively available instrumentation or methods. The majority of PIs should have current NIH grant funding. Proposed projects should be well-defined to the point where a high-dimensional panel is defined. Fluidigm provides over 300 off-the-shelf metal conjugated antibodies, several panel kits, and metal-conjugation kits that can be combined to form the proposed panel. Fluidigm s Maxpar Panel Designer, an interactive web-based design tool, should be used to develop the proposed panels. At least one project should have primary and simple proofof-principle data collected on a CyTOF instrument to demonstrate that the applicant understands sample preparation and processing workflow. The experiment should consist of 1 2 samples stained with a panel of metal-conjugated antibodies developed with support from a Fluidigm field applications specialist. Plan and coordinate your experiments and proposed panels with the Fluidigm sales and applications team several months in advance of the grant deadline. Writing a CyTOF 2 Grant Application 3

4 Technical Expertise Here it is important to demonstrate that the site has sufficient technical expertise (or definite plans to obtain the technical expertise) to ensure successful integration of CyTOF into the facility and the research goals of the community. The placement facility ideally has a strong history of maintenance and administration of a variety of complex instrumentation. Mastery of the mass cytometry workflow includes proficiency in 1) conjugating antibodies to metals using Maxpar labeling kits, 2) multiparameter panel design, 3) standard suspension-based staining techniques, 4) instrument maintenance, calibration and operation, and 5) high-dimensional data analysis, interpretation, and presentation. For core facilities, it is recommended to have an operator with flow cytometry, mass spectroscopy, and/or mass cytometry experience who will become highly trained in the daily operation and maintenance of the CyTOF 2 instrument. It is also highly recommended to involve individuals with bioinformatics experience to help support high-dimensional analysis and interpretation. Proven track record of integrating complex instrumentation into the research community should be highlighted, including training programs and strategies for introducing new technology. Prior experience with validation of breakthrough technology is desirable. Administration This section should clearly describe the structure in place for oversight of the new system and also outline plans for gradual integration of CyTOF into the facility and research community. Because this is a new technology, particular emphasis should be devoted to methods for 1) introducing the technology to the community, 2) engaging new users and projects, and 3) supporting the growth and expertise of maturing users. These activities have the overall aim of generating a growing set of users with proficiency in experimental design, data acquisition, and data analysis sufficient for peerreviewed papers, grant submissions, and oral presentations. Describe your proposed plans for 1) training, 2) outreach, 3) technical support, 4) engaging new experiments/researchers, 5) support of experimental design concepts and analysis, and 6) data management. Any plans for reagent generation should be discussed here. Any plan for regional/collaborative access or outreach is also beneficial to the application. 4 Writing a CyTOF 2 Grant Application

5 Financial Proposed five-year financial plan for the CyTOF system should include expenses for 1) labor, 2) service contract, 3) consumables, 4) argon gas cylinder rental and refill costs, 5) advanced training administered by Fluidigm, 6) analysis software, and 7) data management costs. Funds for any facility renovations to accommodate the instrument should also be identified here. Contact your Fluidigm sales representative for more detailed installation requirements. Projected revenue should be included and is typically derived from hourly rate multiplied by projected use. Institutional Commitment Here it is important to show material institutional commitment to support the success of the CyTOF 2 system, including historical support for other instruments, as well as money budgeted for hiring appropriate FTE, renovations/install requirements, service contract support, etc. It is preferable to show strong institutional support for at least a two-year commitment period. Any statements demonstrating institutional commitment to creating a center of excellence with the CyTOF platform is desirable. For more information, visit fluidigm.com SALES North America info-us@fluidigm.com Europe/EMEA infoeurope@fluidigm.com Japan info-japan@fluidigm.com China Asia infoasia@fluidigm.com Latin America info-latinamerica@fluidigm.com For technical support visit fluidigm.com/support United States and other countries not in Europe or Asia support.northamerica@fluidigm.com Europe support.europe@fluidigm.com Asia techsupportasia@fluidigm.com PHONE United States (toll-free) Press 5 for CyTOF inquiries when operator is logged off Europe Japan China All other countries For Research Use Only. Not for use in diagnostic procedures. Information in this publication is subject to change without notice. Fluidigm, the Fluidigm logo, CyTOF, and Maxpar are trademarks or registered trademarks of Fluidigm Corporation in the US and/or other countries. All other trademarks are the property of their respective owners Fluidigm Corporation. All rights reserved. 12/2014