Flow Cytometry Shared Resource Laboratories
The flow cytometry shared resource laboratories within the College of Medicine are
located in the Medical Sciences Building (MSB 2264, 2228) and the Mitchell Cancer
Institute (MCI 3085). Each of the USA COM FCSRL houses a BD Biosciences FACSAria-SORP
cell sorter and a BD FACSCanto II cell analyzer. The FACSAria-SORP cell sorters are
capable of 12 fluorescence parameters. The sorter has 4 laser lines, including a 60
mW 350 nm UV line, 100 mW 488 nm,
100 mW 561 nm and 40 mW 640 nm lines. The sorters can be used to isolate cells in multiple formats, including 4-way tube sorting and 96-well plate sorting via the Automated Cell Deposition Unit. Usage rates are $45/hour for analysis ($80/hour for users outside the USA system) and $75/hour for cell sorting ($140/hour for users outside the USA system). The BDFACSCanto II cell analyzers are equipped with 3 lasers (30 mW 405 nm, 20 mW 488 nm, and 17 mW 640 nm) capable of 8 fluorescence parameters. The analyzer is available to individual investigators. Appropriate dichroics are available for numerous routine as well as specialized assays for each of the instruments. Usage rates are $25/hour unassisted, $40/hour assisted within the university and $75/hour for ‘outside’ users.
In addition to BD instrumentation, the MCI FCSRL offers the following:
- Nexcelom CeligoS microplate based imaging cytometer
The CeligoS imaging cytometer is designed for performing assays directly in multi-well plates (6-384 wells). There are many advantages to this approach, including lower demand for cells (allowing for the use of rare primary cells isolated from patient samples) and reagents; as well as analyzing the cells in their native state without the need to enzymaticly remove them from their growth substrate, as is required for traditional flow cytometry. It can image cells in Brightfield and 3 fluorescent channels: blue (377/50nm Ex 470/22nm Em)
Green (483/32 Ex 536/40 Em) and orange/red (531/40 Ex 629/53 Em). Using advanced software based image recognition there are over 10 specific analysis modes for a wide variety of applications. The CeligoS is also a self-serve instrument after training is provided. Usage rates are $25/hour unassisted and $40/hour assisted.
- On-Chip Biotechnologies Microfluidic Cell Sorter (3 laser 6 color)
The On-Chip is the world’s first in its class microfluidic cell sorter, developed in Japan. USA MCI was one of the first centers in America to obtain this new technology. Benefits over traditional sorting include the ability to analyze extremely low starting sample volumes/number of cells. The sorting is much gentler and can accommodate a wide range of particles from exosomes to cell spheres using two different size microfluidic chips. The sorter is aerosol free and placed in a BSL2 cabinet allowing for the safe isolation of cells infected with lenti virus or other pathogens. The 3 excitation lasers (405nm, 488nm, and 561nm) are collinear so fluorochromes excited by any laser can be detected in any of these channels: FL1(445/20 nm), FL2(543/22 nm), FL3(591.5/43 nm), FL4(607/36 nm), FL5(676/37 nm), FL6(732/68 nm). User rate is $75/hour assisted only.
- On-Chip Biotechnology SPiS
The SPiS companion instrument does single cell or sphere deposition into 96 or 384 well plates in a far more gentle and reliable manner than traditional sorters.
In addition to BD instrumentation, the MSB FCSRL has the following:
- Agilent Quanteon Flow Cytometer
The Agilent Quanteon cell analyzer was installed in March 2023 and represents the most advanced flow cytometry analyzer technology available. The system is equipped with 4 excitation lasers (405nm, 488nm, 561nm and 635nm) and is capable of 25 simultaneous color detection. It uses SiPM detectors making it one of the most sensitive instruments available. This sensitivity enables the accurate detection of very small particles such as viruses and exosomes in the 100nm size range. The Quanteon is equipped with an advanced sample autoloader, able to sample from a rack of 40ml tubes, or 96 well plates (regular and deep-well) and 384 well plates. Usage rates are $25/hour unassisted, $40/hour assisted within the university and $75/hour for ‘outside’ users.
- The ZetaView® TWIN - NTA Nanoparticle Tracking - Video Microscope PMX-220
The 2-laser platform (488 & 640 nm) was installed in the core lab in May 2019. Nanoparticle Tracking Analysis (NTA) captures the Brownian motion of each particle in the video. Based on the different diffusion movements of large and small particles in the surrounding liquid, the hydrodynamic diameter of the particles is determined. Pattern parameters, such as intensity fluctuations, surface geometry and shape of the particles as well as particle concentration are documented at each recording and can be used to distinguish sub-populations. In addition, the charge state of the particle surface (zeta potential) can be measured via the movement of the
particles in an applied electric field. Depending on the type of sample and the measuring mode, the measuring range is between 15 nm and 5 μm. User rates are $25/hour.
- The Agilent Seahorse XFe24 Analyzer
Installed in the MSB lab in November of 2019, the Seahorse measures the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells in a 24-well plate format. Seahorse XF technology measures the flux of oxygen and protons in the transient microchambers created by specially designed tissue culture microplates in real time. The software calculates the rates of oxygen consumption and extracellular acidification from its measurements of oxygen and proton flux. Usage is currently covered under S10 OD025089-01A1.
- 10x Genomics Chromium Controller
The newest addition to FCSRLs fleet of instrumentation was installed February 2020. The platform uses advanced microfluidics to perform single cell partitioning and barcoding in a matter of minutes. The controller combines a single cell with a single gel bead that is coated with a unique oligonucleotide barcode to form a single cell emulsion droplet, which is referred to as a “GEM” (Gel Bead-in-emulsion). Within each GEM the unique barcoded oligonucleotides are used as primers to generate cDNAs, which can be traced back to cell of origin. The barcoded amplified fragments from hundreds to thousands of cells are pooled to create short read sequencer compatible libraries. Sequencing of these libraries enables analysis of single cells for gene expression, cell surface proteins, immune clonotype, antigen specificity, and chromatin accessibility. There are currently no user fees associated with this system, but advanced scheduling is required.
- Kimtron IC-320
X-ray cabinet irradiation systems are designed to deliver precise X-ray dosages to specimens that range in size from cells to small animals. The Kimtron IC-320 irradiator is available to investigators but requires assistance and training before independent operation is permitted. Applications are varied and include generation of bone marrow chimeric animals, production of non-dividing feeder cells, X-ray virus inactivation, production of sterile insects for reducing insect populations, and X-ray sterilization of items that cannot be steam sterilized.
The MSB facility is operated by Dr. Domenico Spadafora (over 5 years experience in
cytometry) while Steve McClellan (over 30 years cytometry experience) operates the
Dr. Robert Barrington (over 25 years experience) serves as the director of the USA COM FCSRL.