Flow Cytometry

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

Highlights from the NHLBI Flow Cytometry Core (August, 01, 2021 to July, 31st, 2022): Provided cell sorting, cytometry, and immunoassay services (7685.05 hours of services enlisted in table 01 & 02) to 104 Principal Investigators, while maintaining state-of-the-art facility and implementing new technologies. 29 peer-reviewed publications (09 authored; 20 acknowledged). Initiated two 24/7 sites, located in the CRC and Bldg. 10 section, for analytical flow cytometry. The CRC section also provides 24/7, self-operable cell sorting for trained users. Core achieved 98.6% of cost recovery target of FY22. The core received 2 NHLBI Orloff Scientific Awards: o 2022 (with the NHLBI Office of Scientific Director for developing a cost sharing mechanism between NHLBI cores and NIH institutes), o 2022 (with the Laboratory of Mitochondrial Biology and Metabolism for identifying fasting as a nutritional intervention that blunts inflammation). Overview of the NHLBI Flow Cytometry Core Facility Mission statement. The mission of the NHLBI Flow Cytometry Core Facility, established in 2001, is to provide cutting edge services in cell sorting and advanced analytical cytometry to the NHLBI Division of Intramural Research and, by way of a memorandum of understanding (MOU), to NIDDK. These objectives are achieved by acquiring, maintaining, and operating state-of-the-art instrumentation and establishing the expertise required to provide investigators with the resources necessary to accomplish their research goals. Technical expertise, services, and responsibilities. The Flow Cytometry Core has exceptional expertise in flow cytometry and immunoassays. The staff has over 70 years of combined experience in flow cytometry, including experimental design, data interpretation, quality control and set up of complex instrumentation, as well as the advanced analysis of high dimensional flow cytometry data. Services include a broad range of flow cytometric and immunologic assays (Table 1). The Core Facility staff ensures the optimal operation of all instruments, assists investigators in planning and interpreting experiments, trains users to operate instrumentation and perform cytometric assays, and assists in preparing figures and methods descriptions for manuscripts. Key card access for availing analytical cytometry services on a 24/7 basis is provided to trained users. The staff is also responsible for all safety requirements, inventory of supplies, scheduling, billing, and continuing education as needed. Table 1. Summary of services offered by the Flow Cytometry Core Facility Cell sorting. (Index, bulk, 1 6 way). Single cell cloning for RNA & DNA sequencing. Sorting of subcellular particles, such as exosomes and mitochondria. Imaging flow cytometry (up to 12 channels). Nuclear translocation studies. Antigen colocalization studies. Immunophenotyping studies (up to 27 colors). Apoptosis and autophagy studies. Cell signaling assays, such as Phosflow and calcium kinetics. Rare event studies (CECs, CTCs). Phosphoprotein analysis. Cell cycle and ploidy studies. Mitochondrial studies. Functional studies. Preparation of cytospin slides. Immunoassays: Ultrasensitive quantification of low abundant soluble analytes (up to fg/ml) by Quanterix SiMOA. Cytokine and chemokine quantification by Luminex. Extended data analysis and performance of assays. Training of users on instruments and analysis software. Manuscript preparation (methods and figures). Assist PIs / fellows in high dimensional panel design and optimization. As shown in Table 2, the flow cytometry core provides a wide array of cell sorting, flow cytometry, imaging flow cytometry, and immunoassay services, 5 days per week from 8:30 A.M. until 6 P.M. Trained users of analytical flow cytometers have key card access to both 24/7 facilities (all self-operable instruments in the CRC section and all analyzers in the Bldg.10 section). Cell sorting in the Bldg. 10 section is performed exclusively by core staff, while trained users can independently perform sorts using the FACSMelody platform in the 24/7 CRC section. Table 2. Current Core Services and Instrumentation Instrument and location Year Acquired Use and Description Flow Cytometry Symphony A5 Bldg. 10, Rm. 8C104. 2018. Cell analysis, 7 lasers, 30 colors. 24/7 availability. LSR Fortessa Bldg. 10, Rm. 8C104. 2014. Cell analysis, 5 lasers, 18 colors. 24/7 availability. LSR Fortessa-1 CRC, Rm. 5-3209. 2009. Cell analysis, 4 lasers, 15 colors. 24/7 availability. LSR Fortessa-2 CRC, Rm. 3-5225. 2015. Cell analysis, 5 lasers, 17 colors. 24/7 availability. Canto-II CRC, Rm. 3-5225. 2006. Cell analysis, 3 lasers, 8 colors. 24/7 availability. Canto-II Bldg. 10, Rm. N9106. 2005. Cell analysis, 2 lasers, 6 colors. 24/7 availability. Cell Sorting FACSAria 2 SORP Bldg. 10, Rm. 8C104. 2010. 4-way cell sorting, 5 lasers, 18 colors. FACSAria FUSION_S6 Bldg. 10, Rm. 8C104.2020. 6-way cell sorting with biohazard containment, 6 lasers, 30 colors. FACSAria Fusion Bldg. 10, Rm. 8C104. 2015. 4-way cell sorting with biohazard containment, 5 lasers, 18 colors. FACSAria 2 SORP CRC, Rm. 3-5217. 2008. 4-way cell sorting, 5 lasers, 18 colors. FACSMelody CRC, Rm. 3-5209. 2019. 4-way & single cell sorting, 3 lasers, 10 colors. 24/7 availability. Imaging Flow Cytometry Amnis Imagestream Mk II Bldg. 10, Rm. 8C104. 2009. Imaging flow cytometry, 5 lasers, 10 colors. 24/7 availability. Immunoassays Luminex 200 systems Bldg. 10, Rm. 8C104. 2008. Quantitative and multiplexed cytokine measurements. 24/7 availability. Quanterix SIMOA Bldg. 10, Rm. 8C104. Ultrasensitive 3-plex up to fg/ml quantification of proteins in solution. All services, other than training and consultations, are provided on a fee-for-service basis, as per core fee schedule approved by steering committee on yearly basis. Breakdown of core usage in terms of number of PIs during FY22 is listed in Table 3. 104 PIs were provided services by core during FY22. 37% of PIs were from NHLBI, 27% from NIDDK and 36% PIs were from 13 different ICs other than NHLBI and NIDDK. Table 3. core usage in terms of number of PIs. Institute # PI Users NHLBI 39 NIDDK 28 NICHD 12 NCI 5 NEI 4 CC 4 NIA 1 NIAID 3 NICCH 1 NIDCR 2 FDA 1 NINDS 3 NINR 1 Total 104 core achieved 98.6% of revenues against cost recovery target. 73% of revenue was contributed by NHLBI PIs, 13% by NIDDK PIs where as PIs from 13 other ICs contributed 14% of core revenue against cost recovery target. Core served 7685.05 hours of services in FY22 for PIs from 15 different ICs of NIH. Out of 7685.05 hours of services core served 78% of hours for NHLBI PIs during FY22, where as 14% of service hours were towards NIDDK PIs and 8% of hours of services were towards 37 PIs from 13 ICs of NIH other than NHLBI and NIDDK.