Source: MONTANA STATE UNIVERSITY submitted to
MULTI-COLOR FLOW CYTOMETER FOR HIGH-DIMENSIONAL CELL CHARACTERIZATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1031492
Grant No.
2023-70410-41180
Cumulative Award Amt.
$435,645.00
Proposal No.
2023-05379
Multistate No.
(N/A)
Project Start Date
Sep 1, 2023
Project End Date
Aug 31, 2027
Grant Year
2023
Program Code
[EGP]- Equipment Grants Program
Project Director
Bimczok, D.
Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717
Performing Department
(N/A)
Non Technical Summary
This Equipment Grants Project seeks to obtain funding for a Bio-Rad ZE5™ Cell Analyzer, a multi-color flow cytometer for the analysis of cells and small particles. Flow cytometers are research instruments to collect data on a large number of individual cells based on the use of reagentsthat emit fluorescentlight at wavelengths. Flow cytometry has a large number of experimental approaches in the life sciences, including the characterization and quantification of immune cell subsets, collection of functional data on proliferation, death, activation, or cytokine secretion, and analysis of bacteria, viruses and exosomes. The ZE5™ Cell Analyzer is a fully integrated, robust and user friendly flow cytometer for high-throughput, fluorescence-based cell analysis. Specifically, the instrument can measure up to thirty experimental parameters for each individual cell in a cell suspension in parallel, enabling the acquisition of complex, multi-dimensional datasets. The ZE5™ Cell Analyzer will be installed as shared instrumentation in Montana State University's Flow Cytometry Core Facility, where it will be available to trained users from multiple departments in the College of Agriculture and other colleges. The Bio-Rad ZE5™ Cell Analyzer will provide state-of-the-art flow cytometry capacity for research projects in animal and human health, cell biology, and microbiology and will thus enhance fundemantal and applied research and education at Montana State University, Montana's land grant university.
Animal Health Component
40%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113999103020%
3053999109015%
3114099110010%
3114030110110%
1024099110310%
7237010109015%
7234099110010%
7234030110110%
Knowledge Area
311 - Animal Diseases; 102 - Soil, Plant, Water, Nutrient Relationships; 305 - Animal Physiological Processes; 723 - Hazards to Human Health and Safety;

Subject Of Investigation
3999 - Animal research, general; 4099 - Microorganisms, general/other; 7010 - Biological Cell Systems; 4030 - Viruses;

Field Of Science
1090 - Immunology; 1101 - Virology; 1103 - Other microbiology; 1030 - Cellular biology; 1100 - Bacteriology;
Goals / Objectives
High-end flow cytometry equipment is essential to support the research programs of existing faculty and recent new hires, and for attracting excellent new scientists to faculty positions at Montana State University. The goal of this proposal is to obtain a new multi-parameter flow cytometer for high-dimensional analyses, the Bio-Rad ZE5™ Cell Analyzer. Consistent with NIFA's mission to invest in and advance agricultural research, education, and extension, the ZE5™ will advance fundamental and applied research in agriculture and the life sciences by providing state of the art analytical capacity for eukaryotic cells, bacteria, and small particles. The ZE5™ will be integrated into MSU's existing Flow Cytometry Core Facility, maintained by the Department of Microbiology and Cell Biology (MCB). Over the past decades, MSU has made significant investments in the acquisition and maintenance of flow cytometry equipment. However, we are currently lacking instrumentation to perform high-throughput multi-color analyses. Therefore, we request funding for a new Bio-Rad ZE5™ Cell Analyzer, so that we can continue to perform state-of-the art flow cytometry experiments.TheZE5™ combines multiple features that make it an ideal choice for our core facility: (1) compatibility with a wide range of fluorophores from the UV to the infrared range; (2) high-speed data acquisition for up to 100,000 events/sec; (3) a fully integrated, automated sample loader, compatible with all standard tubes and culture plates; (4) a specialized small particle (20 nm to 1µm) analyzer for microbiology, virology and exosome research; (5) optimized detection features for rare cells, (6) an innovative sample pump with minimal dead volume and a recovery option for unused sample; (7) robust design and a user-friendly software; (8) a 4-year extended warranty and remote technical support; and (9) competitive pricing.
Project Methods
The ZE5™ Cell Analyzer will be used as a shared instrument by multiple users to generate primary flow cytometry data from cell or particle suspensions.Flow cytometry is a high throughput method that uses lasers and photometric detectors to analyze fluorescent light emission of cells and small particles. For detection by flow cytometry, cells are labeled with fluorescent-tagged monoclonal antibodies, membrane permeable dyes, fluorescently labeled DNA or RNA probes, or through expression of fluorescent proteins such as green fluorescent protein. In addition, forward and side scatter properties (FSC, SSC) provide information about cell size and granularity. By selecting fluorochromes with unique excitation and emission wavelengths, multiple antibodies and dyes can be combined to generate multi-parameter panels and analyze multiple features in parallel. The ZE5™ Cell Analyzercan analyze up to 27 fluorescence channels simultaneously, in addition to forward and side scatter parameters,enabling multi-parameter analyzes. The Instrument will be made available to all researchers at Montana State University and at other academic institutions in Montana.The ZE5™ generates *.fcs 3.2 output files, the current standard in the field.All users will be strongly encouraged to engage in a two-part data preservation strategy that embraces the benefits of both a disciplinary repository's standards and discrete audience reach (FlowRepository); and a trusted repository's commitment to long-term preservation (Ag Data Commons). This two-part strategy will enable instrument users to meet NIFA Research Terms and Conditions requirements to provide DOIs for research products.The ZE5™ Cell Analyzer will be used for training purposes in ourundergraduate flow cytometry course. This hands-ontraining is a 6-week section of the 4-credit Research Methods in Immunology (BIOM 457R) course. The ZE5™ will enable our undergraduate researchers to gain relevant experience with a modern, high-end flow cytometer. The ZE5™ also will be available to graduate students in the various degree programs at MSU. To introduce our graduate students to flow cytometry, one session of our required Advanced Immunology class (BIO 525) will focus on flow cytometry and its research applications.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:The Cellular Analysis Core facility that manages the ZE5 Cell Analyzer is a research core facility open to all researchers at Montana State University. The target audience for this project are undergraduate and graduate students, postdoctoral fellows, faculty members and staff scientists in the Department of Microbiology and Cell Biology and other life sciences departments. In addition, our core facility is also open to external users. Researchers from Montana institutions that are part of the NIH-funded MT INBRE network can use the Cellular Analysis Core including the ZE5 for reduced user fees. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training in flow cytometry techniques and technology to undergraduate students, graduate students, postdoctoral fellows and other researchers through hands-on and seminar training sessions offered by the instrument manufacturer BioRad and a methods seminar talk by Dr. Bimczok at the Microbiology and Cell Biology seminar series. In addition, MSU research trainees are now using the ZE5 Cell Analyzer for their various research projects and are thus gaining valuable flow cytometry experience with a state-of the art instrument. In particular, MSU trainees have been using the small particle analyzer and developing multi-parameter panels, two applications that were not possible previously. How have the results been disseminated to communities of interest?Following the installation of the BioRad ZE5 Cell Analyzer in November 2023, a training seminar was held that provided information on the instrument. In addition, hands-on user training sessions were also provided by a BioRad representative, which were attended by >15 MSU researchers from six labs. Leading up to the instrument installation and training sessions, PI Dr. Bimczok gave a presentation at the Microbiology and Cell Biology Seminar Series in October 2023, where she provided information on the Cellular Analysis Core Facility, relevant methods, technologies, and applications for flow cytometry, and the specific features and of the new ZE5 Cell Analyzer. This seminar was attended by >30 individuals and enabled us to spread the word about flow cytometry capacity at MSU and particularly the high-throughput, high-parameter ZE5. The ZE5 Cell Analyzer is now listed on the MSU Cellular Analysis Core Website and is available to trained users at all career stages, with new user training provided by Core Manager Mr. A. Sebrell. What do you plan to do during the next reporting period to accomplish the goals?In the next funding period, we will continue to provide routine instrument maintenance and user support for the ZE5 Cell Analyzer, so that we can keep the instrument in good working condition while also supporting increased research output. To increase instrument use, we will further update our website, provide email updates to facility users, and provide information about flow cytometry in internal seminars. We also will include a module on flow cytometry methods in the graduate course in Advanced Immunology, so that graduate students will gain an in depth understanding about flow cytometry technology and applications, and about the instruments including the ZE5 that are currently available at MSU. During the next funding period, we also anticipate that we will approval for our core facility as an official "RED" core facility, which will provide increased financial and administrative support for the core facility from the Vice President for Research and Economic Development Office (VPRED). Our internal application for VPRED approval is currently under consideration. A designation of our core facility as a "RED" core facility will also greatly enhance the visibility of the core, leading to an increased number of users. This will ultimately benefit the current project, as increased facility funding through university funds and user fees will enable us to maintain the instrument, perform repairs if needed, and possibly invest in instrument upgrades such as a robotic plate loader in the future.

Impacts
What was accomplished under these goals? With this instrument award, we purchased a30-parameter, high throughput BioRad ZE5 Cell Analyzer, as described in the proposal. The ZE5 Cell Analyzer is a state-of-the art flow cytometer that features five lasers, 27 fluorescence channels, a small particle analyzer, a universal plate and tube loader, and a robust and user-friendly design. The ZE5 Cell Analyzer was installed in the MSU Cellular Analysis in November 2023, with hands-on and seminar-style training sessions held during the same month. The instrument has been available to internal and external users since then and has been used for 100> hours, with an increasing trend. To make the instrument accessible for MSU researchers, we have updated our core website and have linked the new ZE5 Cell Analyzer to our Cellular Analysis Facility in FluoroFinder, a free online resource that enables scientists to design and optimize antibody panels for specific flow cytometers. In addition to the ZE5 Analyzer, this award also enabled us to purchase a Millipore Ultrapure Water Purification system. This system is now providing clean water to run the ZE5 Cell Analyzer instrument, providing cost savings and contributing to the sustainability of the instrument and our core facility. Overall, the new ZE5 Cell Analyzer has significantly enhanced our flow cytometry capacity at MSU. With this new instrument in place, we have submitted an internal application to obtain approval and support for the Cellular Analysis Core by MSU's Vice President for Research (VPR). This application is currently undergoing review. If approved, our facility will receive financial and administrative support and will be eligible for internal funding opportunities to maintain, upgrade and or repair core instruments including the ZE5. In addition, designation of our core facility as a VPR core will significantly enhance the profile and visibility of our facility at our university. In addition, The Cellular Analysis Core also is a key component of the Research Core for an NIH Center of Biomedical Research Excellence (COBRE) proposal that was submitted in January 2024 and that is currently being considered for funding. If selected for funding, the COBRE will provide support for core facility personnel and for instrument maintenance contracts for up to 15 years.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2024 Citation: B. Tegner Jacobson, Jessica DeWit, Eli T. Selong, McKenna Quirk, Michael Throolin, Chris Corona, Sobha Sonar, LaShae Zanca, Erika R. Schwarz, Diane Bimczok Innovative methodology for antimicrobial susceptibility de-termination in M. bovis biofilms Microorganisms. In revision.
  • Type: Other Status: Other Year Published: 2023 Citation: D. Bimczok. Leveraging the cellular analysis core for high throughput, multi-dimensional cell characterization. MSU Microbiology and Cell Biology Seminar Series, October 2023, Bozeman, MT