The Flow Cytometry Core provides investigators with a resource for analytical and preparative studies of cells using flow cytometry. The Core offers FACS acquisition and analysis, cell sorting, and consultation for experimental design, interpretation and troubleshooting. Applications include immunophenotyping, intracellular staining, molecular and cellular probes such as Ca flux and GFP, CBA (Cytometric Bead Array), DNA cell cycle, TUNEL and Annexin V for apoptosis, sterile sorting for cloning and more. Both the LSRFortessa and Aria II have a green 561nm laser, which is optimal for detection of "mFruit" fluorescent proteins.
New to the Flow Core? (see guide) or
download NewToFlowcore2012.pdf
Analysis workstations and software training are available for researchers. Please schedule appointments for analysis and sorting at 216-445-3798. The Flow Cytometry Core is located in NBb-40. The FACSAria II is located in NE3-224.
The Core offers many services for the analysis or isolation of cells following staining of cellular proteins or DNA with fluorochrome labeled reagents.
StainingCellularDNA2012.pdf )
TunelStaining2012.pdf, Annexin V)
AriaIISortingProcedure2012.pdf )Countess.pdf ) Data is acquired as FCS 3.0 using BD Cell Quest on the FACScans. Additionally, BD FACSDiva Software is used to acquire data on the LSR II. Data can be imported/exported as FCS 2.0 or 3.0 when using FACSDiva. List mode acquisition and storage of data allows for analysis and presentation many different formats according to the needs of the investigator. Data files are permanently stored in personal folders on the LRI servers, readily available to all LRI employees with proper logon accounts. (ServerAccessForm2012.pdf)
CellQuest, FlowJo (FlowJoSiteLicense2012 ) and ModFit software are provided for analysis. Investigators are encouraged to schedule a training session to learn analysis of flow data utilizing one of the available software packages.
All investigators are encouraged to meet with the Flow Cytometry Core technologists to discuss the experiment. This meeting is often useful in that the technical limitations of an experiment are realized and many potential problems can be averted before critical resources are used. The Core can also provide written protocols for staining, fixation and data analysis methods. The Core technologists are available for assistance with data retrieval, analysis and presentation.
Materials available for reference in the Flow Core lab include several textbooks on the basics of flow cytometry and the Current Protocols in Cytometry. In addition, the Core frequently organizes training seminars at different levels of expertise.
The LRI Flow Cytometry Core offers training programs for Beginning and Advanced Flow Cytometer Operators. These courses cover the hardware and software of our analytical instrumentation. It is the belief of the Flow Core that greater understanding of the instruments and physics employed in experiments will lead to dramatically improved data and experimental design. The Flow Core is dedicated to assisting investigators to attain whatever their desired level of expertise.
The Basic course introduces users to the hardware and software of the LSR II or FACScan. It is not meant to immediately provide complete self-sufficiency but rather to embrace the major concepts needed to begin. It is expected that after this training the user will plan and perform their next experiment with the assistance of Core personnel.
The Advanced course provides methods and strategies to optimize high-level multicolor flow cytometry. Material covered is derived from the top thought-leaders in the field of flow cytometry tempered with personal experience and practicality. Building multicolor assays is a process that requires time, dedication and tenacity to overcome hurdles that can appear when attempting to measure many parameters simultaneously. When the investigator can patiently build an experiment with a series of small steps and optimally characterize the instrument settings for their particular research, truly spectacular results
What to Bring
Bring something to take notes on. Samples and supplies will vary greatly based on specific research. Core personnel will help define what is needed on a person-to-person basis.
Investigators must schedule appointments in advance for ALL flow cytometry. Currently, 1-2 days notice is required; however, more notice is necessary for sorting. Viewing privileges of the appointment calendar are attainable through Google Calendar
(Google Calendar.pdf). Please be aware calendar openings do not guarantee an appointment time. Appointments can be made by phone or e-mail. The Flow Core reserves the right to schedule as it sees fit.
The Flow Cytometry Core has acquired a new high performance, fixed alignment cuvette cell sorter that makes sorting easier and more accessible. The Stem Cell Biology and Regenerative Medicine Department courteously funded the Special Order BD FACSAria II. The new sorter improves ease of use, flexibility, and aseptic capability making it the best choice for consistent results in sorting across a broad range of applications.
A BioProtect III Walk In Clean Air and Containment Cabinet houses the FACSAria II. The BioProtect III is a specialty type Class II biological safety enclosure designed to offer personnel, product, and environmental protection from potentially aerosolized biological hazards.
The Special Order BD FACSAria II consists of 488nm, 640nm, 405nm, 561nm and 355nm lasers, providing 16 parameters. The optical filters detect the following fluorophores:
(AriaIIConfig2012.pdf)
The Flow Cytometry Core is now offering the use of the Countess™ automated cell counter. The Countess™ provides fast, easy and accurate cell counting without using a hemocytometer, eliminating the tedium and subjectivity of manual cell counting. Automated counting frees up your time and minimizes subjective judgments that can lead to error. (Countess.pdf)
Scanning is achieved by using a Becton-Dickinson FACScan flow cytometer. The instrument has a highly sensitive laser used exclusively for quantitative analysis. The FACScan is equipped with three detectors to measure fluorochromes that excite at 488 nm. The FACScan is engineered with fixed optical, electronic and fluidic components that cannot be reconfigured.
(FACScan_config.pdf)
Scanning is achieved by using the Becton-Dickinson LSRII flow cytometer. The instrument has highly sensitive lasers used exclusively for quantitative analysis. The LSRII is capable of 12 color and 14 parameter acquisition with 5 lasers (Argon ion 488nm, HeCd UV 355nm, Violet 405nm, HeNe 639nm, and Green 532nm).
(LSRIIConfig2012.pdf )
The Flow Cytometry Core has recently acquired a Special Order BD LSR Fortessa cell analyzer that has the flexibility to support the expanding needs of multicolor flow cytometry assays. The BD LSR Fortessa cell analyzer offers the ultimate in choice for flow cytometry, providing power, performance, and consistency. It is designed to meet the multicolor analysis needs of complex experiments that require a specific combination of unique lasers, detectors, and filters
(FortessaConfig2012.pdf )
Purchase of the instrument was generously made possible by a grant awarded to Dr. Aaron Fleischman (Principal Investigator) in collaboration with Dr. Roy Silverstein, Tom McIntyre, Keith McCrae, Frank Papay, Gary Procop and Rishi Singh to pursue a project entitled "Multi-User BioSensor Integrated Platform”. The goal of the studies proposed is to create a center for business and academic users that will enable research and development of micro-fluidic and nano-fluidic systems and sensors for medical applications. The project is supported by a 3-year, $2.6M equipment grant sponsored by the "Ohio Third Frontier Wright Center for Sensors Systems Engineering”. The project combines capabilities available at the Cleveland Clinic, Case Western Reserve University, Cleveland State University and Ohio businesses to develop a center for the rapid prototyping and testing these systems. Initial focus areas are drug delivery, wound healing, cellular diagnostics, and pathogen detection. Equipment will also be purchased to support rapid prototyping and testing of multi-layer, micro-fluidic and nano-fluidic systems, cell and biomarker characterization, and integration of micro sensors.
Fortessa technical details:
The BD LSRFortessa SORP consists of 488nm, 640nm, 405nm, 561nm and 355nm lasers, providing 20 parameters. The optical filters detect the following fluorophores:
Additional options included:
The High Throughput Sampler (HTS) option is now available in the Flow Cytometry Core. In addition to single tubes this feature allows acquisition of samples from 96- or 384-well microtiter plates. Experiments with large number of samples are becoming increasingly common. The ability to acquire in "batch” mode can increase productivity by allowing investigators the ability to multitask during sample acquisition. The speed of acquisition in high-throughput mode is <15min per 96well plate (2 sec acquisition per sample). Sample carryover from the HTS is <1%.
Before planning an experiment which utilizes HTS acquisition, a meeting with Flow Core personnel is required.
A 15-minute set-up fee will be included for all HTS use.
Current Protocols in Cytometry is available online through the Cleveland Clinic Alumni Library. This resource is reached by searching the library catalog, in the A-Z list by resource name, or in Find Journals.
Lerner Research Institute
Cleveland Clinic, Mail Code NB21
9500 Euclid Avenue
Cleveland, Ohio 44195
