About Laboratory for Cell Analysis

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 two sites within the UCSF community, with cytometers located in Helen Diller Cancer Research Building and Genentech Hall at Mission Bay campus the and the cancer research building on the Mt. Zion campus(CRB). The LCA is supporting over 17 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.

Services

Contact the Core (415.476.2631 or email lca@cc.ucsf.edu) 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 two FACS Calibur benchtop cytometers, one FACSCanto II multi-laser digital cytometer, one FACS Aria II and one FACS Aria III cell sorters. These systems primarily are used 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 is appropriate. For more complex assays requiring more than four differentiating markers, then the high-end multi-laser Canto II system would work well. To rapidly retrieve or sort a particular sub-group of cells from a mixed sample then a cell sorter would be required. Listed below are the different LCA cytometers and their properties:

Click graph to enlarge.

FACS Aria III and Aria II are high-speed digital sorters which incorporate a fixed-alignment cuvette flow cell. Fiber optics direct and focus of 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.

FACSCanto II from Becton Dickinson is designed for advanced analytical research applications using digital signal processing and flow cell optics, similar to the Aria II systems. The Canto II is equipped with the latest technical capabilities - high-speed electronics, multiple laser excitations across a wide spectral range (from 405 up to 640nm) and up to eighteen PMT detectors, and HTS 96 well plate reader.

FACSCalibur 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 Canto II 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.  

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).

BioRad Luminex.  A flexible analyzer based on the principles of flow cytometry enables you to multiplex or simultaneously measure u to 100 analytes in a single microplate allowing for fast and cost effective bioassay results.

Microscopy

The LCA has several digital microscopes available to users, including state-of-the-art laser-scanning and spinning disc confocal microscopes and laser microdissection microscope from Zeiss located at Mt.Zion (Cancer Research Building), and Mission Bay (Genentech Hall and Hellen Diller Cancer Center).

Zeiss Confocal Laser-scanning Microscope LSM 510 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.

Zeiss Cell Observer Spinning Disc Confocal and TIRF Microscope.  The Zeiss cell observer system consists of special objectives, a modular incubation system, highly sensitive camera and piezo focusing devices, the CSU-X1 spinning disc (SD) confocal  unit, and the Total Internal Reflection Fluorescence (TIRF).  This microscope, equipped with a three laser module (405nm, 488nm and 561nm,) is ideal for multichannel high speed imaging in 3D of live cells. In addition, under the TIRF conditions, processes in the border regions between plasma membrane and the cell interior can be examined in great detail and a resolution of < 200 nm in z is achieved by selective illumination.

Zeiss Laser Micro Dissection Microscope.  Zeiss PALM MicroBeam system offers contact free manipulation and microdissection with laser and the separation of the specimen occurs with a laser beam focused precisely and with high accuracy.  This is a tool for isolation of single cells or cell groups in preparation for PCA, RT-PCT, and proteomics.

AxioImagers  and AxioObserver with Axiovision Software. 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.

Nikon Eclipse  Basic upright dual view teaching microscope. 

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.

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.

Zeiss Zen Workstation  Zen software workstation for the post acquisition analysis of microscopy images obtained on the Zeiss microscopes.

This core is supported by a National Cancer Institute Cancer Center Support Grant (5P30CA082103-15). Any publications related to work done by this core, should reference grant number 5P30CA082103-15 and must include a PMCID as required by the NIH. For more information on how to obtain a PMCID, please see http://publicaccess.nih.gov/submit_process.htm.

For more information on the CCSG, please contact Kate Shumate at shumatek@cc.ucsf.edu.