Flow cytometry, typically using fluorescent probes which bind to specific cell associated molecules, allows measurements of various phenotypic, biochemical and molecular characteristics of individual cells (or particles) suspended in a fluid stream. As the cells flow past a focused laser beam of appropriate wavelength, the probes fluoresce and the emitted light is collected and directed to appropriate detectors. These detectors, in turn, translate these light signals into electronic signals proportional to the amount of light collected. Information regarding the relative size and granularity of a cell, for example, is also obtained as these characteristics influence the way in which light is scattered as the cell passes through the laser beam.

The use of flow cytometry at TSRI can be divided into two broad categories, analysis and cell sorting.

Analysis

The ability of flow cytometers to evaluate cells at an extremely rapid rate (e.g. up to 20,000 events per second) makes this technology ideally suited for the reliable and accurate quantitative analysis of selected physical properties of cells of interest. The sensitivity of these instruments for detecting the presence of molecules expressed at low levels is impressive; given high quality cell preparations and reagents, as few as 50 molecules per cell may be detected.

Cell sorting

One of the properties of the larger flow cytometers is the ability to electronically deflect cells with preset, defined properties into a separate collection tube. For cell purification, flow cytometry is especially well suited for applications requiring high purity. Because multiple fluorochromes (e.g. up to eight distinct fluorescent probes reacting with different cell associated molecules) can be assessed simultaneously, cell sorting by flow cytometry can separate complex mixtures of cells on the basis of multiple marker expression.

The Flow Cytometry Core Facility is located on the first floor of the Immunology building and encompasses approximately 900 square feet of space. Additionally, we have a satellite lab in Science Park 3030 Rm. 2030 (please contact staff for more information). The facility is extremely well equipped for both analysis and cell sorting. The Facility is available for use by outside groups on a special basis (please contact the Flow Staff for information as the rates will differ from those posted on the website). A brief description of the cytometers and their particular capabilities is provided below.

Analyzers

1. FACScan. The FACScan, purchased by the institute in l990, is a highly sensitive single laser, nonsorting instrument used exclusively for quantitative analysis applications. Because the FACScan is equipped with three fluorescence detectors, simultaneous analysis of five parameters (two light scatter and three fluorochromes) is routine. FITC, PE and one of the red emitting fluorochromes that excite at 488nm (PI/Red 613/Cychrome/PerCP) can be combined. Unlike the sorters described above, the FACScan is engineered with fixed optical, electronic and fluidic components, giving it the flexibility to function as an investigator-operated instrument.
2. FACS Calibur HTS. The institute has upgraded an investigator operated instrument exclusively for analysis use. This instrument has been modified to optionally utilize a 96-well plate loading format as well as standard tube loading. Additionally, this instrument uses CellQuestPro and PlateManager software designed for Mac OSX.
3. FACS Calibur II. In 2006, to accomdate the increaesed demand for basic 4-color flow applications, a standard two-laser FACSCalibur was moved to Science Park 3030 Rm. 2030. This instrument is available to all users, but is meant to assist those users located across the street from the Core Facility.
   

   The detector arrangement and commonly used fluorescent probes for the FACScan family of cytometers allow for a variety of multiparameter experimental designs:
   

       The flow facility staff maintain these analytical instruments in optimal working order and train investigators in their use but do not operate the instruments for investigators. The hourly charge for TSRI investigators is $50. User Calendars are online and these instruments are available on a first come first served basis. These instruments all have a FileGuard Security System or utilize built-in logiing software. Accounts are obtained from the facility staff.

4. Digital LSR II -1, -2, and -3.   The most advanced benchtop analytical platform utilizes a number of new technological advances: solid state diode lasers, advanced fluorescence detection with fiber optics and optimized beam paths, and the digital signal processing used on the DiVa sorters. These advances combined together on an easy to use benchtop platform will give researchers the means to perform up to 10-color detection utilizing four different excitation sources. For more information go to LSR II Webpage The first of these instruments was purchased in 2003, a second in 2004, and a third in 2006 to accomadate increases user demand

5. Laser Scanning Cytometer.   A multiparameter Laser Scanning Cytometer (LSC) was added to the facility in March, 2000. The CompuCyte LSC bridges the instrumentation gap between high resolution confocal laser scanning microscopy and flow cytometry in that the LSC generates data similar to a flow cytometer, yet can also provide image data since it is microscope based. Unlike a flow cytometry where a single cell suspension is critical to good analysis, the LSC allows suspension cells, adherent cells, and tissue sections to be mounted onto slides and examined. The LSC will be used for applications that cannot be done in flow. For example, the LSC has the ability to automatically and simultaneously measure fluorescence within both the nuclear area and a defined portion of the cytoplasm in order to track movement of proteins such as Cyclin B1 across the nuclear membrane, and can perform automatic counting of fluorescence-in-situ-hybridization probe spots with the simultaneous measurement of cellular DNA.

6. Beckman-Coulter ViCell XR.   This image based hemacytometer was purchased in June 2005 to facilitate viability and cell counts for sorting experiments. Its ease of use has made it suitable as a user operated instrument within the core. For more information go to ViCell XR Webpage

Sorters

1. FACSAria. Our newest sorter, purchased in April 2004, the FACSAria is unique in having the sensitivity of a digital benchtop analyzer with the functionality of the DiVa sorters. Improvements in fluidics and optics have allowed for higher speed sorting (25,000 events/sec at 70psi using a 70um nozzle) with less stress on the sorted cells and enhanced overall sensitivity. The system uses fiber optic guided solid state 488nm and 635nm diode lasers along with fiber guided emission collections (four colors plus scatter off the 488nm and two colors off the 635nm line). These traits allow for an instrument that does not require the extensive alignment optimizations inherent to the jet-in-air sorters making for increased reproducibility in identifying and sorting dim population. For more information see the FACSAria Webpage.
2. FACS Vantage DiVa I . This instrument is one of the most advanced, state of the art flow cytometer currently available. With three laser capability, advanced optics and new generation of mixed gas tunable lasers, this instrument provides enormous flexibility in terms of applications as well as unprecedented sensitivity. The Vantage configurations will be the instrument of choice for the development of new applications, for applications involving dyes requiring nonstandard wavelengths for excitation and for the analysis and sorting of highly complex populations of cells and cells with very low expression of the antigen of interest. The Vantage has been upgraded with the Digital option (DiVa) and many high speed multi-laser sorts of up to four populations simultaneously are now being done routinely with throughputs near 16,000 cells/second and sorted fractions exhibiting > 99% purity upon analysis. Not all experiments are suited for hi-speed sorting, but typically more than half of our murine lymphoid cell sorting experiments are done hi-speed. Cells stimulated to become apoptotic, dendritic cells, and microglia, for example, are too fragile for hi-speed conditions and are sorted under more gentle conditions. This instrument was upgraded from a Vantage SE TSO  in December 2002. The system now has a diode laser specifically set up to use the violet (405nm) wavelength ideal for exciting eCFP.
3. FACS Vantage DiVa II. This system is identical to the exisiting DiVa and was upgraded from a Vantage SE TSO in June 2003.  It currently is configured for detection of up to eight different fluorescent parameters. This systems utilizes a Krypton-ion laser with UV enhanced (351nm) optics as the third laser.  A few examples are shown below:
   

   Examples of DiVa Color Combinations

   FL1	FL2	FL3*	FL4*	FL5	FL6	FL7	FL8
   FITC	PE	Cy5-PE	Cy7-PE	APC	APC-Cy7	PI	Alexa350
   Alexa488	eYFP**	Cy5.5-PE	Cy5.5-PerCP	Cy5		Indo-1	Indo-1
   eGFP		TexasRed-PE		APC-Cy5.5		uvGFP
   Flua-3		Cy5.5-PerCP				CFP**
   		PI
   		7-AAD
   		dsRed

   *There are certain reagent combinations that can be problematic in these channels.  Please consult that facility staff before designing a reagent set.  **Can be used as FRET pairs.

It should be noted that the fluorochromes mentioned above are the dyes most commonly available commercially. New dyes are continually being developed and most of these can be used in this facility. Questions regarding other applications should be directed to the facility staff.

Costs for using the Sorters

    Facility users are billed on an hourly basis using the following scheme (outside users, please contact the Flow Staff for relevant rate information):

    All of the sorters (Vantage, FACStar Plus, etc.) are operated solely by the trained flow facility staff members, with the exception of some analytical experiments where experienced users may run their own assay. To reserve time on these instruments, investigators should contact the flow facility staff at 784-8396 or 784-8286.

    Data analysis is the responsibility of the investigators. The flow facility makes available a variety of workstation and software options for users. The user operated flow cytometers all have Macintosh based computer systems for acquisition, and a Macintosh G3 workstation with CellQuest, ModFit LT, WinList 4.0, FCSPress, FlowJo, Attractors, and a variety of other applications are available for data analysis and desktop publishing. The current rate for the use of the workstation is $3.00/hr, and $0.10 per print job on the Tektronix Phaser 860 color PostScript printer.

We have just added a new G4 workstation suitable for analyzing the complex digital data from the DiVa systems.  This computer is primarily designed for using FlowJo in the OSX (Jaguar) environment.  This system is available during normal hours and the current rate is $5.00/hour.  Please contact the facility staff for more details.

    The Laser Scanning Cytometer runs off a Windows NT system where acquisition and analysis is done using WinCyte software. Flow Cytometry Standard (FCS) data files may also be exported via WinCyte, allowing image files and LSC listmode data to be transferred and viewed with other software packages on other platforms.

    Another cytometry software application, WinMDI, written by Joe Trotter, is available for Windows 95/98/NT systems and is particularly well suited for generating publication quality images. A Dell Dual Pentium 500 running Linux is provided for short term data storage and for networking peripheral laboratory workstations. The Linux server also functions as the facility World Wide Web server on the Internet.

    The technology of flow cytometry is rapidly growing and highly technical. Errors in sample acquisition and/or data analysis are common among poorly trained users and can lead to misleading and/or unreliable data. The facility staff are committed to providing training and education for all the facility users. Group training sessions (at several levels) are available and individual help is provided whenever possible. Contact staff personnel for specific information regarding training sessions. Typically, new TSRI staff that will use the flow cytometry facility are given an orientation (two 1.5 hour sessions) to cover the broad scope of facility use and some specifics on the use of the cytometers. Users that want additional training or people new to flow cytometry are encouraged to contact the director for personal assistance during several data aquisition and analysis sessions before working extensively on their own. Contact the director, for any special assistance or training at 4-8396 or asaluk@scripps.edu .

    Another area of focus also important for assuring high quality work is the dissemination of reliable protocols for specific flow cytometry applications. To this end, a protocol page is under construction on the flow facility World Wide Web server for access over the Internet.

    Finally, one of the major long-term goals of the facility is to collaborate with investigators in the development of new, leading edge applications of flow technology. Investigators are encouraged to discuss their ideas and/or needs with the director of the facility.