Flow Cytometry and Cellular Imaging Core - Smithville
Ellen Richie, Ph.D. and Collene Jeter, Ph.D., Co-Directors
Flow Cytometry Services
Flow cytometry is a fundamental tool used broadly in cell biological research. Specific indicators of cell status and/or protein expression can be measured simultaneously on individual cells within a heterogeneous population. The FCCIC maintains three Becton Dickinson (BD) instruments, two cell sorters and one flow cytometer, all operated using BD FACSDivaTM software. The latest version of this software allows for index sorting, letting researchers identify the origin of single cells collected from within the population defined by their experimental parameters. That is, the specific fluorescence and scatter parameters can be matched to each individual cell that has been sorted.
- Cell Sorting
- Multiparameter Immunofluorescence
- Apoptosis Analysis
- Cell Cycle Analysis
- Single-cell Deposition
- Identification of Side-Population Stem Cells
- Experimental Design and Data Analysis
- Consultation and Training
Flow Cytometry Instrumentation
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 FACSAria™Fusion SORP
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.
BD FACSAria™ Fusion
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.
Cellular Imaging Services
Microscopy imaging and data acquisition have been significantly enhanced by the addition of two new instruments: a Zeiss LSM880 laser scanning confocal microscope with Airyscan technology and a Leica TCS SP8 DIVE multiphoton microscope. The Zeiss instrument provides near-super resolution capabilities, whereas the multiphoton device provides superior imaging of thick sections. The core also maintains a Zeiss Meta 510 wide-field epifluorscence microscope appropriately configured for time-lapse imaging applications. Finally, the core has Bitplane’s Imaris image processing software enabling 3D and 4D image reconstructions and advanced qualitative and quantitative image analysis.
- Image Acquisition
- Protein Distribution Analyses
- Colocalization Studies
- Tissue Architecture Analyses
- UV DNA Damage Experiments
- FRET and FRAP
- Live Cell Imaging
- Consultation and Training
Cellular Imaging Instrumentation
Zeiss LSM880 with Airyscan
Our Zeiss LSM880 confocal microscope is mounted with five standard lasers (a Diode-405 nm laser, an Argon-458/488/514 nm laser, and solid state 561, 594 and 633 nm lasers), a 355 nm DNA damage laser, and an Airyscan detector. Airyscan is a novel photon detector technology with improved signal-to-noise ratio (SNR) relative to conventional GaAsP detectors. Users achieve a 1.7× higher resolution in all spatial dimensions, 140 nm laterally and 400 nm axially. It also has a dedicated incubator chamber for time-lapse imaging and a FRET/FRAP module to analyze dynamic molecular processes. This scope combined with Bitplane's Imaris image processing software will allow for 3D reconstructions of z-stacked images, quantitative analyses, and cell tracking analyses.
Leica TCS SP8 DIVE Multiphoton Microscope
This instrument, equipped with an Insight x3 dual-line infrared laser and 4 HyD detectors, can be used to examine thick sections, intact organs, and in vivo processes with maximal imaging depth, minimal light scattering, increased z-resoution, and reduced phototoxicity. The core also maintains a Leica VT1200S automated vibratome to cut thick tissues and a microdissection fluorescence microscope for tissue preparation. The image to the left is a 3-D tissue reconstruction stained with anti-CD31 (green) and phalloidin (red) to distinguish vasculature and musculature, respectively (bar = 100 µm).
Zeiss Inverted Axio Observer
Our Axio Observer wide-field epifluorescence microscope is equipped with DIC/Nomarksi optics and a full range of objectives (10X, 20X, 40X, 63X and 100X) to meet imaging needs. In addition, it has an incubator chamber, thus making it available for time-lapse imaging applications.
Zeiss LSM880 Confocal Images
Stitched mosaic subset of a 5x5 tile scan showing DNA damage foci - γH2AX (green) and 53BP1 (red) in nuclei (blue).
Multispectral analysis of thymocyte epithelial cells specification during development. Two transcription factors (green and red) specify cell fate.
Tile mosaic of four fields of view showing regenerated prostate cells with basal cell (red) and luminal cell (green) characteristics.
25 fields of view stitched together into a mosaic showing heterogeneity in prostate. Basal cells (red), luminal cells (green), nuclei (blue).
Functional analysis of stem cell markers in colon crypts. Stem cells (green), differentiated Paneth cells (red), cell nuclei (blue).
Analysis of post-translational modification of tubulin in germ cell development. Acetylated tubulin (red), nuclei (blue).
Super-resolution image of a mouse fibroblast cell stained with LAMP1 (green) and LC8 (red) to visualize lysosomes and the dynein motor complex, respectively.
Ellen Richie, Ph.D.
Epigenetics and Molecular Carcinogenesis
Collene Jeter, Ph.D.
Epigenetics and Molecular Carcinogenesis
Flow Cytometry Manager
The Virginia Harris Cockrell Cancer Research Center at the University of Texas MD Anderson Cancer Center, Science Park Department of Epigenetics and Molecular Carcinogenesis
Mailing Address: P.O. Box 389, Smithville, Texas 78957
Physical Address: 1808 Park Road 1C, Smithville, Texas 78957