Flow Cytometry and Immune Monitoring Core
Flow Cytometry and Immune Monitoring Core Facility
The University of Kentucky Flow Cytometry and Immune Monitoring Core Facility offers state-of-the-art flow cytometry cell analysis, high-speed sorting, and immune functional analysis to the research community at UK, and has been in continuous operation since 1983.
Our skilled staff provide a variety of flow cytometric approaches for multiparameter fluorescent analysis and sorting of eukaryotic and prokaryotic cell types and provide a variety of immune monitoring analyses to phenotype immune cell populations and functions. Cell sorting and analysis is also available for live human cells and infectious (BSL-2 level) samples. Training on analytical cytometers is available to investigators and their staff in order for laboratories to analyze their own samples at reduced cost.
Please do not hesitate to contact us if you have any questions about potential uses of flow cytometry for your research projects, how to appropriately design experiments for flow cytometry, prepare samples, or how to analyze your data.
Description of Core for inclusion in “facilities” section of grants
The Flow Cytometry and Immune Monitoring Core (FCIM) provides comprehensive, state-of-the-art analytical flow cytometry cell analysis and high-speed cell sorting services to support both human and animal studies. FCIM has been in continuous operation since 1983 and provides a variety of flow cytometric approaches for multiparameter fluorescent analysis and for the sorting of eukaryotic and prokaryotic cell types. Cell sorting and analysis is also available for live human cells and infectious (BSL-2 level) samples. Training on analytical cytometers is available to investigators and their staff so that individual laboratories can analyze their own samples at reduced cost.
Flow cytometric services include: 1) multiparameter cell phenotyping up to 23 colors on cell analyzers and 12 colors on cell sorters; 2) analysis of DNA content and cell cycle; 3) analysis of cellular apoptosis and necrosis; 4) quantification of cell cytotoxicity; 5) intracellular cytokine and protein analysis; 6) measurement of cell activation and signal transduction parameters; 7) high speed cell sorting of live human and animal cells under BSL-2 biocontainment conditions; and 8) single cell sorting into microtiter plates for cloning or single cell sequencing with next generation sequencing (NGS) technologies on campus.
Immune monitoring services include: peripheral blood cell isolation, plasma/serum isolation, cell banking, cytokine/growth factor analysis (ELISA, Elispot, Luminex) and leukocyte functional assays. In addition, FCIM also offers macrophage/DC generation, T cell expansion and other cellular product and immune assay design services.
Standardized data analysis is available to investigators for each set of samples, depending upon the application. Data can be reanalyzed by staff at the request of the investigator. The core also makes available several workstations and software options for users to analyze their own data at no additional charge. The workstations in the lab have either Mac-based CellQuest analytical software or PC-based Summit software as well as FlowJo software. A site license for FlowJo has been purchased and individual licenses are available to labs for a yearly fee.
Current instrumentation and equipment include the following systems and analyzers.
iCyt-Sony Cell Sorter System: This flow cytometer system contains two separate sorter units housed in a BSL-2 level biosafety cabinet. Each sorter unit is equipped with 5 lasers (355 nm, 405nm, 488nm, 561nm and 642nm) and can sort or analyze cells labeled with up to 12 fluorescent antibodies or probes. In addition, the biocontainment setup allows sorting and analysis of infectious samples (risk groups 1 and 2) and live human samples. Cells can be sorted sterilely into 5 ml (12 x 75 mm) tubes, 15 ml conical tubes or directly into multiwell plates.
Bigfoot spectral cell sorter: The cell sorter with 5 lasers (349 nm, 405 nm, 488 nm, 561 nm, 640 nm) and 53 detectors utilizes the newest technology of spectral flow cytometry, which captures the entire emission spectrum of each fluorochrome rather than just the peak emission band. This sorter is also housed in a BSL-2 level biosafety cabinet for use with infectious samples (risk groups 1 and 2) and live human samples. This sorter can perform 4-way sorting or can be performed into multiwell plates.
Cytek Aurora: The Aurora is full spectrum flow cytometer with 5 lasers (55 nm, 405 nm, 488 nm, 561 nm, and 640 nm) and 67 detectors. The new technology of spectral flow cytometry greatly increases the capacity for multi-dimensional analysis in complex cell populations.
BD FACSymphony (units #1 and #2): The FACSymphony cell analyzers are equipped with 5 lasers and 23 detectors. It can simultaneously measure up to 21 fluorescent antibodies or probes, as well as forward light scatter (cell size) and side light scatter (cell granularity).
CytoFLEX LX (Beckman Coulter): This cell analyzer is equipped with 5 lasers: blue, red, violet, yellow, and near UV and can simultaneously measure up to 19 fluorescent antibodies or probes, as well as forward light scatter (cell size) and side light scatter (cell granularity). It is fitted with plate loader for 96-well plates for analysis, can analyze sample volumes as low as 10 ul and has nanoparticle resolution as low as 200 nm. It also performs absolute cell counts for every sample and population.
MACSQuant VYB (Miltenyi Biotec): This cell analyzer is fitted with 3 lasers (405nm, 488nm and 561nm) which allows simultaneous measurement of up to 8 fluorescent antibodies or probes, as well as forward light scatter (cell size) and side light scatter (cell granularity). This instrument is a fully automated multi-color flow cytometer that performs automated staining of cell samples and automated cell analysis.
AutoMACS Pro Magnetic Cell Separator: This cell sorter is a high performance immunomagnetic cell separation instrument with fully automated functions for hands-free operation, such as fully automated labeling of multiple samples. Sorting on this instrument utilizes antibodies that are labeled with an iron-containing compound, which are then separated automatically from unlabeled cells with a rare earth magnet. Cells can be separated using positive selection, depletion or untouched isolation programs. Cooling racks allow the maintenance of integrity of samples and eluated fractions. This magnetic cell sorter is useful for pre-enrichment prior to cell sorting or analysis, especially when rare cells are of interest.
ImmunoSpot S6 Universal Visible/Fluorescent Analyzer: This instrument provides high-resolution visible and fluorescent light analysis for use in applications that benefit from morphometric analysis, including ELISPOT assays, colony counting (bacterial, yeast, stem cell, tumor cells), plaque and clonogenic assays, and genotoxic/cytotoxic assays. It is compatible with all plate formats from 100mm Petri dish to 384-well microplates.
Data Storage Policy
All flow cytometry data generated by FCIM is stored on a secure university server in a standard FCS format that is readable on any of the common analytical flow cytometry software programs. Researchers and their staff have ready access to any of their data generated over the previous 5 years via this server. The Flow Cytometry data on the network storage is backed up every night by IT Services to tape backups using IBM Tivoli Storage Manager software.