Flow Cytometry
The EMC Flow Cytometry Core, located in South Camus Research Building 4, provides assisted use services (hourly fee) for analytical flow cytometry on all instruments, including the BD LSR Fortessa, BD FACSAria™ Fusion SORP, BD FACSAria™ Fusion and BDAccuri C6PlusTM. Assisted cell sorts are also available on the BD FACSAria™Fusion SORP and BD FACSAria™ Fusion. Instrument training is mandatory for any user of the FCCIC who would perform unassisted flow cytometric analyses. Service fees vary and the fee schedule is available in iLab and below. Please contact Pam Whitney (pjwhitney@mdanderson.org) prior to scheduling initial services.
Services, Applications and Pricing
Analytical Flow Cytometry
Flow cytometry is a fundamental tool used broadly in cell biological research. Protein expression or specific indicators of cell status can be measured simultaneously 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 in developing complex assays to quantitatively examine cell surface and cytoplasmic antigens, cell cycle, apoptosis, "side populations" and fluorescent transgene expression (e.g. GFP, YFP, RFP and BFP).
Cell Sorting
The EMC Flow Core'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 combinations 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, simultaneously.
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.
Sorted Spermatocytes
Other Services
- Multi-parameter analysis and sorting
- Full-spectrum analysis of fluorescent proteins
- autoMACSpro available for depletion/enrichment of cell populations prior to flow experiments
- Sorting cells into a variety of tubes and plates including 96- and 384-well plates
- Index sorting allowing identification of which experimental population a particular cell was derived
- Guidance with experimental design
- Consultation and training
Training
The core recommends first-time users contact Pam Whitney (pjwhitney@mdanderson.org) to schedule a consultation before finalizing any experimental plans. This consultation will provide input on the best experimental approach, the most ideal fluorescent panels and the 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 one week prior to the date of service. Planning in advance will allow researchers to have an increased selection of times on their chosen instrument.
Instrument training is mandatory for any user of the flow core who would perform unassisted flow cytometric analyses. Training is provided by Pam Whitney (pjwhitney@mdanderson.org) and will encompass blocks of times over multiple days.
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 all users are responsible for archiving their own data. Core personnel can assist users in learning this practice.
Pricing
| Services | Instruments | Assisted Rate | Independent Rate | Unit |
|---|---|---|---|---|
| Multi Parameter Analytical Flow Cytometry | BD LSR FortessaTM | $65.61 | $38.67 | Per Hour |
| Cell Sorting | BD FACSAriaTM Fusion SORP | $75.39 | $57.98 | Per Hour |
| Cell Sorting | BD FACSAriaTM Fusion | $76.21 | $60.32 | Per Hour |
Instrumentation
BD FACSAria™Fusion SORP
The BD FACSAria™Fusion SORP is a 5-laser, 18-parameter, high-speed cell sorter, with automatic cell-dispensing unit, 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 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 cell-dispensing unit on this instrument can dispense single cells into 96- or 384-well plates. The sample chamber and collection apparatus are climate-controlled and aerosol-contained. A laser configuration file for this instrument may be downloaded here.
BD FACSAria™ Fusion
The BD FACSAria™ Fusion is a 5-laser, 17-parameter high-speed cell sorter with biosafety cabinet and automatic cell-dispensing unit. 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 functional analyses. This instrument provides exceptional sensitivity and resolution for optimal multicolor analysis and cell sorting. It allows 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. A laser configuration file for this instrument may be downloaded here.
BD LSRFortessa™
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. A laser configuration file for this instrument may be downloaded here.
Miltenyi autoMACS® Pro Separator
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
BD Accuri C6Plus™ Cell Analyzer
The core's newest instrument is a BD Accuri C6PlusTM 2 laser, 4-parameter cell analyzer that is compact and easy to use. It is optimized for fluorchromes such as FITC, PE, PerCP-Cy™5.5, APC, and BD Horizon Brilliant™ Blue 515 and can analyze many variants of fluorescent proteins, such as GFP, YFP, and mCherry.
Links and Resources
Sample Preparation
Samples
- 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.
Controls
- 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 Pam Whitney (pjwhitney@mdanderson.org) for more information about the requirements for your experiment.