The FCCIC provides assisted use services (hourly fee) for analytical flow cytometry on all instruments, including the BD LSR Fortessa, BD FACSAria™ Fusion SORP and BD FACSAria™ Fusion. Assisted cell sorts are also available on the BD FACSAria™Fusion SORP and BD FACSAria™ Fusion. Training and unassisted use (at a discounted rate) are available for all instruments. Instrument training is mandatory for any user of the FCCIC who would perform unassisted flow cytometric analyses or sorts. Service fees vary and the fee schedule is available in iLab along with our Service Request Form. Please contact Pam Whitney (email@example.com) prior to scheduling initial services
Services and Applications
Flow cytometry is a fundamental tool used broadly in cell biological research. Protein expression or specific indicators of cell status can be measured simultaneoulsy for individual cells within a larger, heterogeneous population. The core provides access to both to advanced instruments and expert staff to meet most any researcher need. The core specializes indeveloping complex assays using their BD LSR Fortessa 15 parameter cell analyzer to quantitatively examine cell surface and cytoplasmic antigens, cell cycle, aopotosis, "side populations," and fluorecent transgene expression (e.g. GFP, YFP, RFP, and BFP. In addition to this key instrument, our FACSAriaTM instruments can also be used for more reoutine flow analysis applications.
The FCCIC's cell sorting equipment includes a BD FACSAria™ Fusion, 17 parameter, high-speed cell sorter for processing of ≥ BSL2 samples within a biosafety cabinet, and a BD FACSAria™ SORP, 18 parameter, high-speed cell sorter. The core can isolate a wide variety of different cell types based on many different combination of antibody-based stains, fluorescent protein expression, and viability indicators. Cells are detected using the BD FACSDiva™ software and can be collected from a single heterogeneous sample into 1-4 tubes, simultaneoulsy.
The latest software version allows for index sorting, letting researchers identify the origin of single cells collected from within the population defined by their experimental parameters. The automatic cell-dispensing unit can dispense single cells into 96- or 384-well plates. The sample chamber and collection apparatus are climate controlled and aerosol contained.
The core also operates a Miltenyi Biotec autoMACS Pro Cell Separator, an immunomagnetic cell separation system that can be used to enrich or deplete a population of cells prior to sorting on one of our BD instruments.
- Multi-parameter analysis and sorting on all instruments
- autoMACSpro available for depletion/enrichment of cell populations prior to flow experiments
- Laser configuration allows for full spectrum analysis of fluorescent proteins and core personnel routinely execute experiments with 10-12 colors
- Sorting into a variety of tubes and plates
- Index sorting
- Guidance with experimental design
- Instrument training for independent users
- Training in applying and using FlowJo analysis software
The Flow Core recommends first time users contact Pam Whitney (firstname.lastname@example.org) to schedule a consultation before finalizing any experimental plans. This consultation will provide input on the best experimental approach, the most ideal fluroescent panels, and design of specific assays to ensure recovery of meaningful data as well as user expectations and cost of services for the scope of the project.
The Core recommends that users schedule appointments at least 1 week prior to the date of service. Planning in advance will allow researchers to have an increased selection of times on the instrument of their choice.
Instrument training is mandatory for any user of the Flow Core who would perform unassisted flow cytometric analyses or sorts. Training is provided by Pam Whitney (email@example.com) and will encompass blocks of times over multiple days. After completing training, a certificate of completion will be awarded.
Core personnel can also assist with data analysis using FlowJo software, or they can perform analysis for a specific project, as time permits, for an additional fee.
Please note that each user is responsible for archiving his or her own data. Core personnel can assist users in learning this practice.
The BD FACSAria™Fusion SORP is a 18-parameter, high-speed cell sorter, run by core personnel. This instrument provides researchers with the capacity to isolate and capture viable cell subsets or single cells for molecular, genetic and/or functional analyses. This 5-laser instrument can distinguish many fluorescent proteins, stem cell and side populations, in addition to fluorescent antibody preparations. Cells are detected by digital BD FACSDiva™ software and collected into one or as many as four tubes simultaneously from a single sample. The automatic cell-dispensing unit on this instrument is able to dispense single cells into 96- or 384-well plates. The sample chamber and collection apparatus are climate-controlled and aerosol-contained.
The BD FACSAria™ Fusion is a 16-parameter high-speed cell sorter. It is the primary flow core instrument and is run by core personnel. It provides researchers with the capacity to isolate and capture viable cell subsets or single cells for molecular, genetic and/or functional analyses. This instrument provides exceptional sensitivity and resolution for optimal multicolor analysis and cell sorting. Its 4-lasers allow users to distinguish many fluorescent proteins, fluorescent antibody preparations, and other markers to identify stem cell and side populations. Cells are detected by digital BD FACSDiva™ software and collected into one, or as many as four tubes, simultaneously. The automatic cell-dispensing unit deposits single cells into 96 or 384 well plates. The enhanced stream stability increases sorting purity and efficiency to help isolate minor subsets of cells (e.g. stem cells) for subsequent in vitro and in vivo experiments. This instrument is contained in a bio-safety cabinet permitting analysis of human specimens while ensuring user protection.
The BD LSRFortessa™ is a 15-parameter cell analyzer. This is a 4-laser instrument equipped with digital BD FACSDiva™ software. Capabilities provided by this instrument include quantification of cellular subsets based on the expression of surface or intracellular antigens, analysis of apoptosis and cell cycle parameters, and multiparameter analyses. The core offers researchers individualized training on the BD LSRFortessa™ for unassisted operation.
The autoMACS® Pro Separator by Miltenyi is an automated high-speed immunomagnetic cell separation system with the ability to process multiple samples at a time. Many cell types from several species and tissue types, including whole blood, can be processed with the autoMACS® Pro with the use of immunomagnetic beads. Cells can be separated using positive selection, depletion or untouched isolation programs. It can sort more than 10 million cells per second from samples of up to 4×109 total cells. The instrument has a touch screen and easily navigable menus. The system reduces manual handling of samples and increases reproducibility
Links and Resources
- All samples should be at a concentration of 5.0 x 106 cells/ml in appropriate buffer in 12 x 75 FACS tubes or 15 ml conical tubes depending upon instrument.
- Bring additional buffer to the core for concentration adjustments.
- All samples must be filtered before running on the instruments.
- Each experiment must have proper controls. Biological controls for data samples could include isotype controls for antibody staining or stained normal cycling cells for cell cycle analysis.
- The instrument setup requires additional controls: unstained cells and single-stained color controls.
- Contact the FCCIC for more information about the requirements for your experiment.