Flow Cytometry and Immune Monitoring Core

Flow Cytometry and Immune Monitoring Core

The Flow Cytometry and Immune Monitoring (FCIM) core has been in operation since 1983 as a fee-for-service core facility to provide access to state-of-the-art equipment, as well as technical and scientific expertise for comprehensive analysis of immune responses. The FCIM Core occupies 1,290 square feet of space in the Healthy Kentucky Research Building (HKRB076) with a fully functional wet lab, shared equipment area, and office space. The Core has two faculty members, Dr. Jamie Sturgill and Dr. Siva Gandhapudi, who serve as co-directors to oversee day-to-day operations and research activities and three full-time, PhD-trained staff members to provide scientific and technical support for the core users.

The FCIM core operates in a BSL-2 plus environment. It offers a variety of flow cytometric services, including flow cytometry training, flow cytometry panel design/optimization, high parameter cell phenotyping using flow cytometry, technical assistance in flow data collection/data analysis, and high-speed cell sorting. The core also offers comprehensive immune monitoring services, including human peripheral blood cell isolation, plasma/serum isolation, cell biobanking, multiplex protein/ analyte detection using Luminex or Isoplexis technology, functional immune profiling at the single-cell level using Isoplexis, antigen-specific T cell detection assays (ELISPOT assay, AIM assay, and ICS assay), real-time cell metabolic analysis using Seahorse technology and custom immune assay design service.     

FCIM core houses several state-of-the-art shared equipment which is available 24-7 for all trained users at the University of Kentucky. The current instrumentation available are listed below:

Flow cytometers

Cytek® Aurora Spectral Analyzer: The Cytek® Aurora spectral flow cytometer is a high-parameter flow cytometer system with five lasers (355nm, 405nm, 488nm, 561nm, and 640nm) and 64 detectors (16 in UV (UV1-16), 16 in Violet (V1-V16), 14 in Blue (B1-B14), 10 in Yellow-green (YG1-YG10), and 8 in Red (R1-R8)) to capture light scatter and emission wavelengths. The detection modules consist of avalanche photodiodes (APDs) coupled with narrow bandpass filters (~15-30nm wide) ranging from 355-830nm.

BD FACSymphony (2 units available): The FACSymphony A3 cell analyzers are equipped with 5 lasers (355nm, 405nm, 488nm, 561nm, and 637nm) 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 (350nm, 405nm, 488nm, 561nm, and 638nm) 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 a 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.

Cell sorters/enrichment units

BD Symphony S6 Cell sorter: The FACSymphony A6 cell sorter I is enclosed in a BSL II safety cabinet and is equipped with 5 lasers (355nm, 405nm, 488nm, 561nm, and 637nm) and 23 detectors. It can perform 6-way sorting and temperature control modules. It can perform 16 gates deep sorting hierarchy and is fitted with nozzles in size ranging between 70-130µm.

autoMACS® Pro 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 the 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.

Analyzers

ImmunoSpot S6 Universal Visible/Fluorescent Analyzer (CTL): 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.

IsoSpark Analyzer (Bruker): IsoSpark, designed by Isoplexes, is a fully automated chip-based technology that performs functional immune profiling at the single-cell level. Using a micro-fluidics platform, the chip-based assay is used to detect secreted and intracellular proteins or protein phosphorylation at a single-cell level without further cell manipulations.  

xMAP INTELLIFLEX ® System (Luminex): A compact flow-based multiplex bead-based platform equipped with 532nm green laser and can accommodate 96 and 384 well plates. The instrument can run procartea plex kits for multiplex protein-based immune assay and Invitrogen QuantiGene multiplex gene expression systems. The instrument is capable of detecting up to 400 analytes.

Cytation 5 Cell imaging multimode reader (Agilent): This instrument has an automated microscopy and microplate reader platform fitted with the multimode detection modules, including filter- and monochromator-based fluorescence detection, luminescence, and UV-Vis absorbance detection systems.

Seahorse XF Pro Analyzer (Agilent): Seahorse XF Pro analyzer measures and reports the oxygen consumption rate (OCR), Proton efflux rate (PER), or extracellular acidification rate (ECAR) and ATP production rates on cells and organoids in a 96-well format. This is a fully automated system that performs compound addition and mixing, label-free analytical detection, and automatic measurements in real-time. 

Note: As of 5/16/2024 this section is under evaluation and revision, and will be updated shortly.