The Laboratory for Cell Analysis provides cytometric support for the UCSF Helen Diller Family Comprehensive Cancer Center. The LCA was established in 1983 with the following responsibilities:
- Provide flow and image cytometers and the technical support needed to make use of these instruments.
- Educate UCSF students, staff, and faculty in cytometry technology and applications.
- Develop new cytometric methods and new cytometric applications.
The LCA currently occupies four sites within the UCSF community, with cytometers located in the cancer research building on the Mt. Zion campus, Clinical Sciences and Health Science West on the Parnassus campus, Building 1A on the SFGH campus, and Genentech Hall at Mission Bay. The LCA is supporting over 30 instruments representing a long-term capital investment of over $7M.
The LCA was one of the first large cytometry cores in the country and has developed a broad repertoire of instrumentation, technical expertise, and a thoughtful infrastructure to help address a wide variety of experimental demands and novel applications in many model systems. From apoptosis to zymogen assays in organisms from adipocytes to zebrafish, the LCA has accumulated working protocols and a broad theoretical knowledge base to aid Cancer Center investigators.
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Services
Contact the Core (415.476.2631 or email) to discuss your research needs and access to LCA services. Core personnel will help you decide which protocols and instrumentation will best suit your needs.
Flow Cytometry, Cell Analysis/Sorting
The LCA provides access and training on one Accuri C6 and four FACS Calibur benchtop cytometers, four LSRII multi-laser digital cytometers, three FACS Aria and one FACS Aria II cell sorters. These systems primarily are for the rapid analysis and sorting of single-cell suspensions. To analyze the light scatter and fluorescence characteristics of a cell population, one of the benchtop machines should be appropriate. For more complex assays requiring more than four differentiating markers, then the high-end multi-laser LSRII systems would work well. To rapidly retrieve or sort a particular sub-group of cells from a mixed sample then a cell sorter would be requested. All LCA flow systems have specialized software for data processing, cell cycle modeling by DNA content, and batch processing of sample data. Listed below are the different LCA cytometers and their properties:
FACS Aria and Aria II are high-speed digital sorters which incorporate a fixed-alignment cuvette flow cell. Fiber optics direct and focus three or four lasers (488nm, 633nm, 561nm, and 407nm or 355nm), depending on which system, onto the alignment prisms, and then focused on the cuvette flow cell. The flow cell allows for improved light excitation and collection optics. The collection optics consist of octagon- and trigon-shaped collection devices, and a total of up to 18 independent signals can be acquired at one time on a single-cell basis, followed by purification of cell subsets by sorting into tubes or multiwell formats.
FACS LSRII from Becton Dickinson is designed for advanced analytical research applications using digital signal processing and flow cell optics, similar to the Aria systems. The LSRIIs are equipped with the latest technical capabilities - high-speed electronics, multiple laser excitations across a wide spectral range (from 405 up to 676nm) and up to eighteen PMT detectors.
FACSCalibur (Mt.Zion, Parnassus, and Mission Bay) is a benchtop flow cytometer designed for applications ranging from routine clinical to basic research. The system analyzes cells as they pass one at a time through two focused laser beams, a 488nm argon-ion laser and a 635nm red-diode laser. The FACSCalibur system is simpler than the LSRII measuring forward light scatter (FCS), side scatter (SSC), and four fluorescence parameters as well as the pulse area and width of any fluorescence parameter and time. The FACSCalibur on Parnassus is equipped with a 40-tube autosampler carousel for walk-away acquisition and analysis. This instrument also has a fluidic sort module for isolating or sorting subpopulations.
Accuri C6 Flow Cytometer is the newest benchtop flow cytometer designed for applications similar to the FACSCablibur but packaged in an all digital electronic instrument. The all digital format means there is no more complex setting of PMT voltages or dealing with logarithmic analog amplifiers. In addition, the C6 includes volumetric sampling so the system can count cells per ml or measure precise volumes (e.g., stop acquisition at 100ul).
Microscopy
The LCA has several digital microscopes available to users, including new state-of-the-art laser-scanning confocal microscopes from Zeiss located at Mt.Zion (cancer research building), Parnassus (HSW), and Mission Bay (Genentech Hall).
Zeiss Confocal Laser-scanning Microscope LSM 510 NLO and LSM510 META. These microscopes come with four confocal channels for reflected light, plus one for transmitted light. Each channel has individual 12-bit analog-to digital converters to ensure optimum data acquisition. The LSM 510 uses Digital Signal Processor to control data acquisition, scanner operation, and the acousto-optical tunable filters (AOTFs), and it has the capability to change the settings for lasers and detectors very quickly. AOTF is used for tuning the power of each laser line and image acquisition, which means multiple regions of any shape can be selected, scanned with specified laser power, and detected with optimized settings. Another unique feature is the META spectral detection system, which gives the emission spectra at each pixel. This spectral signature facilitates the deconvolution of overlapping dyes and probes. Specialized software for 3D image processing is available for image analysis, including automated measuring functions for the quantitative analysis of 3D and 4D (i.e., time) data sets.
Leica Laser Micro Dissection Microscope. The Leica LMD microscope combines an automated upright microscope, a three-dimensional optical control of the dissection laser beam, and non-contact sample collection by gravity. This is a tool for isolation of single cells or cell groups in preparation for PCA, RT-PCT, and proteomics.
Zeiss Axioplan with Axiocam and Axiovision Software and Leica microscope with openlab. These are general-use, universal microscopes, for use in all areas of light microscopy, including transmitted and incident light microscopy, fluorescent microscopy, darkfield, phase, and polarization contrast.
PowerGene MacProbe and MacKtype Systems is a digital microscopy system for capturing fluorescence in situ hybridization (FISH) images and karyotyping. It has optimized optical filters, CCD camera, and powerful image manipulation and analysis tools. MacProbe offers multi-channel image capture, comparative genomic hybridization (CGH), fiber FISH, and a general image measurement package. The MacKtype karyotyping system offers automated karyotyping process from its one-click auto capture capabilities to its comprehensive chromosome analysis package.
Timelapse Equipment. All systems are based upon imaging of samples in 6- or 24-well plates, placed on a movable stage housed within a temperature- and CO2-controlled environment. Acquisition and analysis software (Openlab, from Improvision) allows saving of data as Quicktime movies that are compatible with most presentation applications. The systems for phase contrast timelapse videomicroscopy include these:
Zeiss Axiovert 100 microscopes, equipped with 5 x , 10 x and 20 x objectives, and COHU video cameras are used to examine cell morphology, particularly the processes of cell death and cell division. Samples can be studied in 6- or 24-well plates, and experiments typically run for several days.
One advanced system for microinjection, phase contrast, and fluorescent timelapse videomicroscopy.
One Zeiss Axiovert TV100 microscope, equipped with a Hamamatsu ORCA 100 CCD camera, objectives from 5 x to 40 x, an Eppendorf microinjector arm, and a mercury arc lamp and adjustable excitation and emission filter wheels. This system allows for combined phase contrast and fluorescent imaging of up to 3 fluorophores. Fluorescent imaging allows study of transfected populations, of intracellular localisation of various EGFP-based fusion proteins, or of other markers of organelle function (e.g. mitochondrial membrane potential reporter dyes). Samples can be microinjected and recipient cells studied for precise kinetic analyses.
Data Analysis
The LCA provides several options for post-acquisition flow and image data processing, quantifying results, and presentation. How your data was acquired will determine which option to choose.
ModFit LT from Verity Software House is used when analyzing DNA content and cell cycle by flow cytometry. It offers rapid, accurate analysis of DNA data files with report-ready screen layout, flexible databasing, and user analysis setting.
FlowJo from Tree Star is designed for viewing and analyzing flow data. It provides a very fast method of post-acquisition manipulation of sample data that can be applied to all samples in a data set instantly. Advanced data presentation tools are available. Post-acquisition, multi-channel compensation for spectral spillover between parameters is also available.
Openlab from Improvision is designed specifically for scientific imaging applications. It consists of centralized Shared Resources and an extensive range of modules. The Shared Resources include a comprehensive series of functions for image handling, enhancement, processing, measurement, calibration, presentation, animation, and publication. Additional modules are available for specific functions and applications such as removal of out-of-focus haze or ratio images from different wavelengths.
IVIS Living Image is for use in quantifying and analyzing data obtained from the Xenogen murine imaging system.
Zeiss LSM Examiner is for post-acquisition analysis and manipulation of images obtained from the Zeiss LSM Confocal Laser-scanning Microscope.
Improvision Volocity provides users with an interactive volume visualization system that will run on a standard desktop computer. Volume interactivity provides users with an enhanced perception of depth and realism. Volocity is a true-color 4D rendering system designed for biomedical imaging. It uses advanced algorithms to provide high-speed, easy-to-use, interactive rendering of time-resolved color 3D volumes. Volocity allows the user to visualize a 3D object and then observe and interact with it over time, and it provides users with a system to dynamically visualize both the structure and the purpose of biological structures.
Education
The LCA offers introductory courses on flow cytometry, image cytometry, and use of the analysis programs.
A quarterly Flow Cytometry Course is a beginning course that explains in some detail what flow cytometry is and how to use the instruments available in the LCA. Participants are eligible for two hours of free cytometer use with a lab member in order to familiarize them with how to use the cytometers for their own experiments. The course is free for all Cancer Center members and their staff. (See Recharge Rates for cost to non-members.)
A quarterly Image Cytometery Course covers the fundamental theories of image cytometry and applications for various lab procedures. It provides an introduction to all the image cytometric instruments available at the LCA. The course is free for all Cancer Center members and their staff. (See Recharge Rates for cost to non-members.)
Training is provided on all LCA instruments on an individual basis at the "with-operator" rates.
Additional Resources
A searchable database of core facilities at all UCSF campus locations, provided by the Clinical and Translational Science Institute at UCSF, is available here.
