Iowa State University Service Facilities for Biotechnology ...
Service Facilities for Biotechnology Research
Iowa State University
A major initiative of Iowa State University has been the establishment and support of state-of-the-art instrumentation for biotechnology research. The Iowa State University Instrumentation Facilities for Biotechnology Research are located on the university’s campus in Ames, Iowa. The facilities are open to faculty and students from the university, other educational institutions and industry scientists.
In addition to instrumentation facilities, the university has in place an organization whose primary function is the support of researchers’ equipment needs. The Research Equipment Assistance Program (REAP) maximizes use of research and teaching equipment by maintaining an equipment location database and loan program through which interdepartmental loaning and sharing of equipment is conducted. The REAP office has in inventory various pieces of research equipment that are available for loan.
The following are brief descriptions of the services offered by each of the instrumentation facilities. For additional information, see biotech.iastate.edu/service_facilities.
Animal Gene Transfer Facility
The facility provides a variety of services to individuals who wish to utilize transgenic animals as part of their research program. The facility maintains equipment necessary for the creation of transgenic animals via microinjection or somatic cell nuclear transfer. The facility is available to life science researchers for technical service and for training of a limited number of individuals. All work to be performed in this facility must be scheduled with the professor-in-charge, as the facility is not staffed full-time.
Equipment housed in the facility includes two microinjection workstations, micropipette pullers, a microforge, stereo-zoom microscope and CO2 incubator. Additional supporting equipment located in nearby laboratories to which access may be granted includes a heated microcentrifuge, tri-gas incubators, stereo-zoom microscopes, inverted microscope, biosafety cabinet, osmometer, pH meter, embryo freezing machine, cell fusion machine, gel electrophoresis equipment and UV transilluminator.
Atmospheric Air Quality Laboratory
The laboratory is situated at the National Swine Research and Information Center and serves as a focal point for collaborative research and training in the area of air quality engineering and livestock odor. The laboratory specializes in continuous air quality monitoring, environmental analysis, quantification of organic compounds with gas chromatography and mass spectrometry, and simultaneous chemical and olfactometry analyses.
Field Air Quality Measurements
The laboratory is equipped with a variety of real-time analyzers for gaseous ammonia, hydrogen sulfide, carbon dioxide, methane, volatile organic compounds, particulate matter and ozone. It is also equipped to conduct field air sampling and analysis of organic gasses with sorbent tubes and SPME.
Multidimensional Gas Chromatograph-Mass Spectrometry-Olfactometry (MDGC-MS-O)
The MDGC-MS-O system includes custom-made GC-MS system with heat-cutting capability based on a Dean switch capable of simultaneous chemical and olfactometry analyses.
Solid Phase Microextraction (SPME)
SPME is used for extractions of gases emitted from a variety of samples, including livestock and poultry manure, air, food, packaging, bio-based products, live insects, plants, breath and general applications related to quality control and quality assurance.
Atomic Force Microscopy Facility
Three atomic force microscopes are available for use in the Roy J. Carver Laboratory for Ultrahigh Resolution Biological Microscopy of the Institute for Combinatorial Discovery.
Digital Instruments DimensionTM 3000 Scanning Probe Microscope
The Dimension 3000 scanning probe microscope (SPM) brings together all SPM techniques in a single platform and handles a wide range of sample sizes and types. A rigid, low vibration construction of the Dimension 3000 SPM ensures the highest quality images and measurements.
Samples up to eight inches in diameter can be scanned in ambient air or fluids using the Dimension 3000 SPM. The Dimension 3000 SPM requires little or no sample preparation, and the simple vacuum mounting system allows easy and convenient setup. Superior linearity and resolution in all three dimensions are obtained, even for large samples. Integrated top-view video optics with motorized zoom and 1.5 µm optical resolution help identify areas of interest for detailed scanning quickly and easily. Changing scanning techniques, for example from AFM to STM, requires no tools.
The NanoScope IIIa system controller is a main part of the SPM system providing the software and electronics that drive the microscope. Digital tracking and feedback control ensure accuracy and speed at all scan sizes and positions on the sample.
Digital Instruments MultiMode with a Tapping ModeTM
The MultiMode system features multiple scanners that permit the user to tailor the system for individual research. Scanners with large scan ranges up to 120 microns on the X–Y axes, and a Z range up to 6 microns, as well as high-resolution scanners with 0.5 micron X–Y axes and submicron Z range are available. The vertical-engage “JV” and “EV” scanners allow the tip to be positioned at any point on the surface, without adjusting for lateral movement during approach. The MultiMode is controlled with a NanoScope IIIa controller. This controller provides 16-bit resolution on all three axes, with three independent 16-bit digital-to-analog converters (DACs) in X and Y for control of the scan pattern, scaling and offset. This configuration provides 16-bit resolution of the lateral scanning motion at any scan size.
Digital Instruments DimensionTM 3100 Scanning Probe Microscope
The Dimension 3100 is controlled with a Nanoscope IV controller. The NanoScope IV features up to ten-times-faster scanning, as well as increased functionality, bandwidth, flexibility and expandability.
Center for Crops Utilization Research
The Center for Crops Utilization Research (CCUR) is a resource to assist ISU researchers and off-campus businesses in developing new value-added processes, products and markets for Midwest crops, especially corn and soybeans. CCUR strives to add value to grain and other crop-derived materials by conducting grant and contract research; offering short courses, workshops, seminars and training experiences; providing analytical, pilot plant processing, and consumer evaluation services; providing technical consulting services and information retrieval; and operating small-business incubator services. The center has grain, food and material processing equipment in state-of-the-art laboratory and pilot plant facilities.
The pilot plant facilities include a 5,000 square-foot wet-processing pilot plant (soy protein isolation, corn wet milling, brewing, etc.); a 2,600-square-foot dry-processing pilot plant (dry corn milling, drying, grinding, sieving, etc.); a 900-square-foot hazardous solvents extraction facility (vegetable oil extraction grain, plant material extractions); 3,000-square-foot product development laboratory (plastic extrusion, molding, and film blowing; building material processing; etc.); and various process development and analysis laboratories (chromatography, grain analysis, vegetable oil refining, baking, laboratory-scale process development, etc.). A small theater and conference facilities are available for technology transfer activities.
CCUR partly administers and works closely with the ISU Fermentation Facility, the Iowa Grain Quality Initiative and the Grain Quality Laboratory.
Chemical Instrumentation Facility
The Chemical Instrumentation Facility has more than five million dollars worth of analytical instrumentation available to faculty, graduate students, industry and other educational institutions. The staff of five highly qualified professionals supports university research by keeping the analytical equipment available and operable and by providing application support and user training. Services available to the research community include the following:
Magnetic Resonance
Eight NMR spectrometers of varying frequencies from 60 to 600 MHz are available. Magnetic resonance spectroscopy allows the use of atomic nuclei as magnetic probes within a molecule. Chemical analysis and spatial orientation are determined by using this technique. A fully equipped EPR system also is available. Equipment located in the facility includes a Bruker Avance 600 (solids) NMR, a Bruker DRX-400 NMR, a Varian VXR-300 NMR, a Varian VXR-400 NMR, a Bruker AC-200 NMR, two Varian EM-360 NMRs and a Bruker ER-200 EPR. A Bruker Avance 700 MHz NMR is available in the Molecular Biology Building.
Mass Spectrometry
The mass spectrometry lab is equipped to provide both low- and high-resolution GC-MS on mixtures and high-resolution measurements for determining the elemental composition of pure samples. In addition, electrospray and APCI are used for the ionization of higher molecular weight compounds, particularly those of biological origin. Two dedicated LC-MS are now available for special projects. MS-MS (parent-daughter relationship) experiments are routine. A fully equipped MALDI-TOF for the analysis of biopolymers also is available. Equipment located in the facility includes a Finnigan TSQ-700 GC-LC-MS, a Kratos MS-50 MS, a Finnigan Magnum ITD GC-MS, a Micromass GCT-MS, a Shimadzu LCMS2010, a Finnegan LCQ LCMS and a Bruker PROFLEX-DE MALDI-TOF.
X-Ray Diffraction
A completely equipped X-ray diffraction laboratory provides instrumentation for the study of the molecular structures of small molecules and powders. The equipment includes a Bruker APEX II CCD single-crystal diffractometer equipped with low-temperature devices and a Scintag SDS-2000 powder diffractometer available for general use.
Spectrophotometry
A variety of spectrophotometers is available for routine use. These instruments provide “fingerprint” spectra for characterizing and identifying compounds. These instruments currently include a Bruker IFS 66V FT-IR, a Hewlett-Packard HP-8452 Diode Array UV-Vis and a Jasco J-710 circular dichroism spectrophotometer.
Elemental Analysis
A Perkin-Elmer Model 2400 Series II CHN/S elemental analyzer is available for sample submission or for investigator use. Normally, the instrument is configured for carbon, hydrogen and nitrogen, but sulfur also can be analyzed upon special request.
Computation
In addition to computer systems associated with the instrumentation, numerous PCs and workstations are available for network-based data processing and modeling. All data acquired in the facility are automatically backed up to tape or CD.
Confocal Microscopy Facility
The Confocal Microscopy Facilities of the Office of Biotechnology, the Plant Sciences Institute and the Institute for Combinatorial Discovery have three confocal microscopes available for use by on- and off-campus researchers.
The confocal microscopes are located in 0117 Molecular Biology Building, the Roy J. Carver Co-Laboratory and the Roy J. Carver Laboratory for Ultrahigh Resolution Biological Microscopy. Researchers can choose which microscope best fits their research needs.
Confocal microscopes remove out-of-focus fluorescent light from the image, allowing clearer imaging of the sample, including the ability to view structures and components that were obscured by excess fluorescence that would be generated by standard fluorescence microscopes. Some applications of confocal microscopy include fluorescence microscopy when spatial distribution of cellular or other structures is important to the research being conducted, acquisition of a stack of images that can be used for 3D-reconstruction, viewing structures in thick tissue and removing background and cross-talk fluorescence or fluorescence from other structures that obscure viewing of the desired structure.
Leica SP5 X Confocal Microscope in the Molecular Biology Building
The confocal microscope in the Molecular Biology Building allows for real-time optical sectioning of fixed and living specimens, providing significant improvements in optical contrast and resolution over traditional light and fluorescence microscopy. The facility is equipped with a Leica SP5 X confocal microscope system with inverted microscope front end. New technology available on this system includes a white light laser. Researchers are no longer limited to three or four lasers at three or four fixed wavelengths but can tune the white light laser to any wavelength between 470 and 670 nm, which has a similar effect to having 200 lasers and 200 wavelengths. This allows researchers to select the excitation wavelength that is best suited to their samples and will result in maximum fluorescence emission. On the emission side, the system has an Acousto-Optical Beam Splitter (AOBS) which allows researchers to precisely set the emission wavelengths they would like to capture rather than be limited to preset emission ranges determined by fixed filter sets. Both of these technologies help to separate fluorescence signals with close excitation and/or emission spectra.
Other features include faster scanning, higher resolution and increased sensitivity. Researchers will now be able to work with live cells over an extended period using live cell equipment including a heated stage with microcontainment system, active gas regulation, cell cultivation chamber and microinjection system. For capturing images of fast moving samples or events that occur in a fraction of a second, the resonant scanner enables video rate scanning with speeds up to 16,000 lines per second. In addition, the system has FRET, FRAP, timelapse, hyperspectral signal separation software, 3D imaging, colocalization, deconvolution, region of interest (ROI) scanning, brightfield and DIC capabilities. A 405 laser provides UV excitation and an Argon laser provides CFP excitation and extra power for bleaching. The system also has an X-Y scanning stage and automatic composition software to capture high resolution images of samples that are too large to fit within one field of view and a color digital camera.
Nikon C1si Confocal Microscope in the Roy J. Carver Co-Laboratory
The facility at the Plant Sciences Institute is equipped with a Nikon C1si confocal microscope with both standard fluorescence confocal detection and spectral imaging capabilities. The 440, Argon, 561, and 638 lasers offer excitation wavelengths of 440, 457, 476, 488, 514, and 638 nm. Capture of transmitted light images is also available. The system offers 40x and 60x water dipping objectives in addition to 10x dry, 20x dry, 60x water, and 100x oil objectives. While in standard confocal detection mode, the system can capture up to three fluorescence channels and one transmitted light channel simultaneously. Additional capabilities include time lapse, FRET and FRAP. The system’s spectral imaging mode is useful for separating closely overlapping emission wavelengths from multiple fluorescent probes and/or autofluorescence. In spectral imaging mode, the system can simultaneously acquire up to 32 channels (2.5nm, 5nm, or 10nm individual channel widths) of fluorescence spectra in a single pass. Additional capabilities in spectral imaging mode include FRET and time lapse. A digital camera also is available on the system for standard (non-confocal) microscopy images.
Prairie Technologies Scanning Confocal Microscope in the Roy J. Carver Laboratory for Ultrahigh Resolution Biological Microscopy
A Prairie Technologies scanning confocal microscope is part of an optical workstation attached to an inverted Nikon Eclipse 200 microscope. Excitation wavelengths of 488, 568 and 633 nm are provided by Argon, Argon/Krypton and HeNe lasers. The instrument is completely computer controlled, including choice of dichroic mirrors, filters, pinhole size, scan size, integration time, photomultiplier voltage and z-focus. Images are stored on a hard drive and are available for export via Internet and CD. Image software is available that allows 3D reconstruction of confocal images generated with the confocal microscope.
DNA Facility
The DNA Facility of the Office of Biotechnology performs DNA synthesis, DNA sequencing, high-throughput DNA sequencing, plant genomic and plasmid DNA extraction, automated fluorescent genotyping, quantitative, real-time PCR and automated microarray slide hybridization.
DNA Sequencing
Sequencing samples are run on the DNA Facility’s Applied Biosystems 3730xl DNA Analyzer. The AB 3730xl uses a four-color dye system and provides up to 900 bases of usable sequence data per reaction. DNA can be sequenced as plasmid, lambda, cosmid or BAC DNA, or as PCR products (direct sequencing). Custom primers can be used with all types of templates. Clients submit sequencing orders using the OnCore software. Use of this software allows clients to track the progress of their orders and will automatically notify them when their data are ready to download. A four-color printout of the data is provided. When no problems are encountered with a template, the results are generally returned within 24-48 hours after receipt of the samples. The facility also provides a primer walking service.
High-Throughput DNA Sequencing
For clients who have high-throughput sequencing projects, samples can be submitted in 96-well format. The Applied Biosystems 3730xl is capable of processing twelve sets of 96 samples in a 24-hour period. Users need to contact the facility before submitting samples for high-throughput projects to obtain information regarding sample submission, etc.
Massively Parallel (Next Gen) Sequencing
Access to both short- and long-read massively parallel sequencing instruments is made possible through an instrument-sharing agreement between Iowa State University and the University of Iowa. The Illumina Genome Analyzer II operated by the ISU DNA Facility generates millions of short-reads (75 bases per read). Its applications include ChIP-seq, digital gene expression tag profiling, whole genome and candidate gene re-sequencing, and small RNA identification and profiling. The University of Iowa DNA Facility operates a Roche 454 Genome Sequencer FLX that is capable of long-read sequencing (400 bases) and is especially well-suited for de novo sequencing of new genomes for which no reference sequence exists.
Both facilities provide library construction services for their respective instruments but also accept client-prepared libraries. The appropriate facility should be contacted before submitting samples to obtain information regarding sample submission requirements, data handling and pricing.
DNA Template Preparation
The facility performs plant genomic DNA preparation using the AutoGen 740 instrument. Plasmid template preparation in 96-well format also is available. In addition, the facility offers a seed grinding service using its Spex Certiprep GenoGrinder.
Automated Genotyping
The facility processes primarily microsatellite and AFLP markers using an Applied Biosystems 3730 DNA Analyzer to electrophorese samples and collect the gel image. Each sample can have as many markers as the client can identify. The data are analyzed, and automated allele calling of microsatellites can be performed by the ABI Genomapper software. Electronic files are provided via the facility server.
DNA Synthesis
The DNA Synthesis Service synthesizes DNA oligomers in two scales, 50-nmol and 200-nmol, and can make modified oligomers such as the fluorescent primers used in genotyping applications as the client desires. In addition, primer design for primer walking sequencing projects is available for those who desire this service. Oligos are synthesized using a BioAutomation MerMade-192 DNA synthesizer.
Quantitative Real-Time PCR
The DNA Facility has available two quantitative, real-time PCR instruments – the Stratagene Mx4000 and the Stratagene Mx3005 – and will accept jobs on a ready-to-run basis. Applications include gene expression studies, validation of microarray data, allelic discrimination, SNP analysis, and screening for GMOs. DNA staff are also available to advise or assist clients at any point in the experimental process from initial project design through chemistry and material selection and data analysis.
Microarray Slide Services
Slide Hybridization – An Amersham Lucidea SlidePro hybridization unit is available for use by on-campus users. This instrument can perform hybridization and washing of up to six microarray slides in a uniform and highly-reproducible manner.
Slide Scanning – Microarray slides are scanned using Applied Precision’s array WoRxe® Biochip Reader. It is a high-resolution, white light, CCD-based system that provides high quality images with accurate and reproducible results. Slides can be analyzed using API’s SoftWoRx Tracker.
For related services, please see Genomic Technologies Facility and GeneChip® Facility.
Doubled Haploid Facility
The development of homozygous lines is an important, but time-consuming, process in plant breeding and research. The induction and subsequent doubling of haploids is an efficient alternative to generate homozygous offspring in two generations.
The Doubled Haploid Facility (DHF) provides haploid doubling for maize to ISU scientists, as well as of-campus academic researchers and breeders. The start-up phase is supported by the Department of Agronomy which provides use of its laboratories, greenhouses and nursery.
The mission of DHF is to:
• provide expertise and service in the production of doubled haploid lines in maize
• improve the technology in order to get higher success rates and lower costs
• teach and train scientists and students
The production of doubled haploid lines consists of three main steps:
1. haploid induction by pollination with inducer lines,
2. selection for haploid kernels, and
3. doubling of haploid plants and subsequent seed production.
Haploid Induction
For haploid induction, maternal inducer genotypes, such as RWS described by Roeber et al. 2005 are used. In the facility’s experiments, it finds an average induction rate (IR) of ca. 6% in diverse plant materials.
Haploid Kernel Selection
Kernels with a haploid or F1 embryo can be distinguished by means of the expression of the dominant anthocyanin marker gene R1-nj. The expression of this gene provides an anthocyan pigmentation of the embryo and the endosperm. Kernels with a pigmented endosperm and a nonpigmented embryo are selected as haploids.
Doubling
Spontanous chromosome doubling occurs but is dependent on the genotype. To obtain more consistent doubling rates, plants are treated with colchicine following the procedure (method II) described by Eder et al. 2002. Doubling rates (DR) of 20-30% are realized. A success rate of 14% was obtained in preliminary experiments since not all doubled plants will produce pollen and can be selfed.
The production of the double haploid lines requires two generations. For one generation, the facility uses a winter nursery.
Environmental Engineering Research Laboratory
The laboratory; a service of the civil, construction and environmental engineering department; provides chemical analysis and related training and consultation services in support of university-sponsored research. Documented quality control receives top priority and is made available to researchers wishing to verify the quality of results.
Facility staff can function as consultants to assist the ISU research community by preparing quality assurance plans for research proposals, configuring computer systems for data acquisition and manipulation, training departmental personnel in analytical instrumentation and methodology, implementing chemical hygiene plans and preparing specifications for instrument purchases. Most of the laboratory’s major instrument systems are available for use by researchers who wish to do their own analytical work. The laboratory provides training and supervision for those researchers.
Atomic Absorption Spectrophotometry
Flame atomic absorption and emission are available. A 150-position autosampler and a GBC 932 Plus Atomic Absorption Spectrophotometer can be used to determine metals in large numbers of samples.
Automated Analysis
A four-channel Technicon AutoAnalyzer II Industrial System is used to automate nearly any colorimetric test. The facility custom-builds AutoAnalyzer manifolds for specialized research applications.
A Seal Analytical AQ2+ automated discrete analyzer is available for unattended automated analysis of nitrogen, phosphorus, silica, sulfate, chloride, and cyanide.
A BD-40 block digester is available for simultaneous, semi-automated Kjeldahl nitrogen and total phosphorus digestions. Data collection, computation and reporting are carried out with a microcomputer data station.
Mercury Analysis
A Leeman Labs Hydra AF Gold Plus automated mercury analyzer is available to analyze for trace levels of mercury. The analyzer uses cold vapor atomic fluorescence with dual stage gold amalgamation and dual detectors.
GC/MS
A Varian Saturn 2100 ion trap GC/MS system that includes MS/MS, SIS, and CI capabilities is available. The system includes an autosampler and GC/MS data station.
Gas Chromatography
Four gas chromatographs served by EZ Chrom Elite Chromatography Data Systems provide a wide range of options for organic analysis. FID, ECD, TCD, ELCD, PID and NPD detectors are available. Autosamplers are available for liquid injection as well as purge-and-trap sampling. Chromatographs are equipped for operation with packed, capillary or Megabore columns. Sample preparation techniques include continuous or discrete liquid-liquid extraction for water samples and sonication or Soxhlet extraction for soil, tissue and other solid samples.
Carbon Analysis
A Shimadzu TOC-Vws TOC Analyzer is available to analyze carbon in liquid samples by the UV-promoted persulfate oxidation method.
Other
The laboratory is equipped for most types of wet chemical analysis, including related spectrophotometric and potentiometric methods. Membrane filter techniques are used for bacterial testing.
Fermentation Facility
The Fermentation Facility of the Office of Biotechnology, Center for Crops Utilization Research and chemical engineering and food science and human nutrition departments offers services, including the production of microbial cells and their metabolites. The facility is rated for BL1 containment.
Pilot-scale equipment housed in the Food Sciences Building includes two B. Braun fermentors with working volumes of 100 and 50 liters, two NBS fermentors with working volumes of 115 and 22 liters and one Bioengineering fermentor with a working volume of 15 liters. Benchtop fermentors located in the Food Sciences Building include two NBS Microferm fermentors (6 or 12 liter working volumes), four NBS Bioflo 310 fermentors (1.2 or 5 liter working volumes) and three B. Braun Biostat M fermentors (2 liter working volumes). Benchtop fermentors located in Sweeney Hall include two NBS Bioflo 110 units with 1 and 5 liter working volumes.
Downstream processing equipment includes an Amicon DC10-L ultrafiltration system, BioSpec BeadBeater, CEPA Z-41 continuous centrifuge, SLM “French” pressure cell press and a VirTis Ultra 35, eight-shelf freeze dryer with stoppering capability.
Fitness and Metabolism Unit
The Fitness and Metabolism Unit (FMU) is designed to facilitate research projects involving human subjects, featuring a variety of research, laboratory space and equipment for controlled studies that involve diet, exercise and behavior change. Equipment may be used by researchers to collect data on metabolic risk factors, body composition and/or physical fitness. An exercise area with private locker room and shower facilities provides convenient space for controlled exercise studies. Equipment includes a treadmill and three cycle ergometers.
Individual testing rooms are available to conduct a variety of clinical measurements. Two rooms are set up with metabolic carts and can be used for assessing resting and exercise-related energy expenditure. Another room is designated for bone densitometry via dual-energy x-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT), with an adjacent room available for body composition assessment using bioelectrical impedance analysis (BIA) and anthropometry. Two phlebotomy rooms are available for collecting blood and other clinical data. The FMU has a fully equipped metabolic kitchen, with a professional oven, microwave ovens, two dishwashers, refrigerator/freezer, walk-in pantry, walk-in cold room and freezer and an ice machine. Adjacent dining rooms provide capacity to serve meals for up to 40 people at one time. Additionally, the on-campus facility has the capacity to provide meals for 24 participants simultaneously, as well as analytical laboratories for blood, urine and fecal sample analyses. The FMU has a private room for clinical counseling/interviewing, as well as computers equipped with software available for data processing and dietary intake analysis. The statistics graduate assistant uses SAS to perform statistical analyses.
Flow Cytometry Facility
The Flow Cytometry Facility of the Office of Biotechnology offers flow cytometric analysis and cell sorting for a wide range of research applications. Facility personnel are trained to assist researchers in flow cytometry experimental design, sample preparation techniques and data analysis. Training sessions to provide general instruction in these areas are also offered by the facility. All Flow Cytometry Facility services are open to Iowa State University clients, as well as to off-campus institutions and individuals.
Flow cytometry has been used to analyze bacteria, various mammalian cell types, fungi, yeast, Drosophila cells, soybean cyst nematode eggs, Euglena, Tetrahymena, dinoflagelletes, plant cells, nuclei, organelles and chromosomes. Immunofluorescence measurements are often used to provide information on lymphocyte subsets and cell surface receptor densities. DNA/RNA-specific stains supply information on genome size, chromatin structure and cell cycle kinetics. Fluorochromes are available for quantitating intracellular pH or cytoplasmic-free calcium. Phagocytosis of fluorescently labeled particles (beads, yeast or bacteria) can be quantitated. Cell viability can be measured for mammalian cells, as well as bacteria. Fluorescent lipophilic dyes are available that have been utilized as a means of tracking cell life and tissue localization in vivo. Levels of enzyme marker gene expression, such as ß-galactosidase, can be correlated with the fluorescence intensity of cleaved substrate by flow cytometry. Intracellular protein products can be measured by immunofluorescent labeling of fixed cells. Fluorescence in situ hybridization (FISH) techniques provide information on the mRNA expression level of a specific gene and can be used in conjunction with flow cytometry to provide quantitative gene expression information on a cell-by-cell basis. Necrotic versus apoptotic-mediated cell death can be distinguished using flow cytometry. It is also possible to sort individual cell populations via flow cytometry, enabling researchers to separate and further characterize subpopulations of cells.
Flow Cytometry Data Acquisition and Cell Sorting
The Flow Cytometry Facility maintains three flow cytometers: a Becton-Dickinson FACSCanto, Becton-Dickinson FACScan and Guava Technologies Personal Cell Analyzer (PCA). The facility also maintains several computer workstations with software packages for performing off-line analysis of flow cytometry data.
BD FACSCanto
The FACSCanto flow cytometer combines a patented optical design and manufacturer upgrades for simultaneous data acquisition of eight fluorescent signals (FITC, PE, PE-TxRed, PerCP-Cy5.5, PE-Cy7, APC, Alexa 700 and APC-Cy7) and two scatter parameters (FSC and SSC), digital electronics for processing up to 10,000 events per second, and a novel sample injection system supporting carryover of less than 0.1%. High-speed data processing, industry-leading sensitivity ( ................
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