The Laboratory of Flow Cytometry has extensive and advanced facilities. The laboratory is located in and occupies most of the 4th floor of the research building immediately adjacent to the hospital. With its prime location proximal to the departments that require the critical informartion that the Flowcytometry laboratory provides, the laboratory is in excellent position to provide timely, critical, and life saving support and information to the physicians that work at RPCI. The laboratory also has extensive contracts to carry out work with other groups that are located outside the hospital in the greater Buffalo area. The equipment that comprises up the backbone of the laboratory is described in detail below.
Instrumentation
In our experience, there are three kinds of scientists who use flow cytometry: those who are already proficient in flow cytometry and who already know its capabilities; those who use flow cytometry as a part of their research program, but depend on the expertise of the flow cytometry staff for providing reliable service; and those who have just learned about flow cytometry or who are just considering the application of this technology to their research problems. We must serve all three of these user groups efficiently, and to do so, we train principal investigators and their technical staff to perform their own flow cytometry. In 2000 we trained over 100 individuals in 12 separate training sessions. This is in addition to personalized consultation services in which flow cytometry staff spend 20 –30 hours per week with over 100 users of the facility. These individuals became proficient in sample preparation, immunophenotyping and DNA content measurements. They learned how to operate a flow cytometer and to analyze and interpret the acquired data. We charge $35.00 per hour for instrument acquisition time, but data analysis is free. The facility is subsidized by a Cancer Center Research Support Grant from NCI.
Our staff, rather than the user, performs all cell-sorting experiments due to the complexity of the instrumentation. We also charge $35.00 per hour for this service. The addition of high speed sorting capability has allowed us to increase our capacity. The reduced sort time has been met with increased user satisfaction because they get their product much faster.
FACSVantage Turbosort:
This flowcytometer is capable of high-speed cell interrogation and sorting. The electronics of the instrument have recently been redesigned for pulse process speeds in excess of 2 MHz. This increased speed is reflected in viable cell recoveries in excess of 80% at sorting speeds of 20K/sec (faster sorting rates provide increasing frequencies of dead cells). This instrument has several other modifications to provide advanced flow cytometry applications.
Forward Scatter Detectors:
These include both forward angle scatter and forward angle fluorescence detection (FFD). FFD is a new parameter that may be useful in providing additional cell resolving power.
Side Scatter Detectors:
A standard side scatter detector at 488 nM excitation is provided.
Seven Color Detection:
Standard flow cytometry instrumentation provides up to six measurement parameters and two computed parameters for a total of eight parameters. The six measured parameters usually consist of FSC, SSC and four colors of fluorescence excited by one or two laser beams. Using current acquisition software up to nine real time parameters can be acquired. Using three spatially separated lasers (UV, 488, 560 - 640 nM tunable dye lasers) with three independent timing windows up to seven colors can be simultaneously measured. Using the primary beam for 488 nM excitation four colors can be measured; two colors for secondary beam excitation and one color for tertiary laser excitation. This provides exceptional flexibility for combining antibody measurement with other fluorochrome combinations such as DNA dyes or for other fluorochrome measurements.
The Laser Window:
We have found that coaxial beams are not practical for resolving most fluorochromes with differing excitation spectra. This problem is completely solved by using separate laser windows so that excitation/emission occur at different times. This allows for resolution of fluorescence based on different excitation wavelengths even if the emission spectra are identical.
High Speed Sorting:
There are two paradigms that need to be clearly defined when we speak of high speed sorting. The first is “high speed analysis” and slow speed sorting. This actually is the most common form referred to as high speed sorting. In reality, however, this is not high speed because the cells to be sorted are infrequent (often less that 5%) and only conventional sorting speeds are required. For example, if 1% of cells were desired, an analysis speed of 30,000 cells/sec. would result in a sorting speed of only 300 cells/sec. This paradigm is actually high-speed cell analysis to increase throughput but only conventional sorting speeds are required. This is very important for stem cells and mixed populations in which the cell of interest represents only a small fraction of the total. The second paradigm is truly high speed sorting, as the events to be sorted are a significant fraction of the total (e.g. 50%). In this situation both high-speed analysis and sorting are required because an analysis speed of 30,000 cells/sec. would require a sorting speed of 15,000/sec. The FACSVantage with TurboSort Option was conceived to meet these design features. A sort rate of 30,000 cells/sec. (1.08 x 108 cells/hr) at 97% purity are routine.
Four-Color FACScan:
A modified FACScan is available to perform simultaneous four-color data acquisition using a single laser excitation source. Usually the four fluorochromes used for antibody conjugation are fluorescein isothiocyanate (FITC), phycoerythrin (PE), PE-Texas Red (PE-TR) and PE-Cy5 or perdinin chlorophyll protein (PerCP). The FACScan has the capability of measuring only three of these colors. In order to accomplish four color measurements in 1993, two major modifications were made to a standard FACScan. In the first, a fifth photomultiplier tube (PMT) and associated electronics were added including the necessary compensation networks. This was wholly designed and built in our electronics laboratory and provides an example of our capabilities in instrument design and modification. In the second, the fixed filtration holders were changed to interchangeable filter holders. This provides us the flexibility of changing filters and dichroic mirrors.
During the design of the compensation networks, we addressed the problem of high autofluorescence associated with many cultured cell lines. This problem can be completely solved by electronic autofluorescence reduction on a cell by cell basis (Cytometry 7:566-574, 1986, Communications in Clinical Cytometry, 22:250-255, 1995). This FACScan allows for changeable optical filtration and one can use one PMT for optimal detection of the desired fluorochrome and a second PMT that accepts the more broadly distributed longer wavelength autofluorescence emission that is mainly outside the fluorochrome’s emission spectrum. The longer wavelength autofluorescence emission is subtracted from the shorter autofluorescence emission thereby nullifying autofluorescence. Since reduction is on a cell by cell basis, when the fluorochrome is present its fluorescence is detected with optimal efficiency regardless of the cell’s autofluorescence.
Three-Color FACScan:
This is a standard instrument used for measuring FSC, SSC and three colors of fluorescence.
FACSCalibur:
This two laser, 6 parameter, 4 color instrument provides both clinical research and reference laboratory service, but is also available for use by research investigators. The autoloader provides for completely automated sample data acquisition.
In March 2000, RPCI purchased a Luminex-100 to upgrade the Resource’s ability to meet the growing demand for multiplexed immunoassays of cytokines. This two laser instrument is used for multiplexed analysis of various bioassays using microbeads in a 96 well microtiter plate system. Up to 100 analytes per well can be screened simultaneously. The system includes the Luminex 100 reader, the XY platform and the SD sheath fluid delivery system. Currently, the Luminex 100 is used for multiplex screening of cytokines and chemokines but it can be expanded for other formats including nuclei acid assays, receptor-ligand assays, enzymatic assays and other immnuoassays.
Computers
The instruments all use Power PCs. Off-line PCs, Macs and even HPs are available for data acquisition. Data analysis is accomplished off line using either MACs or PCs
Accounting Program
We only charge for data acquisition, analysis of data is free. A user is issued a password on the computer attached to the instrument, which keeps a record of time for the session.
Network File Access
In order to accommodate the increasing number of users we have optimized their throughput by providing file access over a computer network. Since data acquisition is usually quite rapid but data analysis can be time consuming, especially when more complex multiparameter analysis is performed, we have always analyzed data off-line. We also do not charge users for analysis time, so they can take as long as desired to evaluate their data. It has been our goal to allow the investigators to perform these analyses in the convenience of their own laboratory. They can access their files over the institute network or by “sneakernet”, using zip drives or CD “burner”.
Computer Software
The facility has available, for its users, nearly all of the current software used to analyze data. This availability is primarily the result of the facility Director’s interest in new approaches and the concomitant beta testing provided by our staff. Facility staff and investigators are currently using the following data analysis packages.
BD Biosciences:
Cellquest:
This data acquisition and analysis program operates in the Macintosh environment. This program is both a data acquisition and analysis program.
Verity Software House:
Winlist:
This multiparameter data analysis program incorporates a fast and efficient data analysis engine with extensive graphics capability for viewing the data. Because of the Dynamic Data Exchange capability of Windows, spread sheet and data base programs can be run in the computer background for instant data exchange with WinList. Because our users have PCs this is by far our most popular program. This program is also available for the MAC environment.
Modfit:
This program is designed to analyze DNA histograms and it has advanced capabilities for aiding the user in handling background, aggregates and debris. The program automatically links to WinList so that combining immunophenotyping with DNA content analysis can take place simultaneously. This program is also available for the MAC environment.
Phoenix Flow:
Multicycle
This DNA analysis program is similar to Modfit in capabilities. This PC based program is also popular among users and some use both to compare results. This comparison can be informative when data quality is poor because each program uses somewhat different algorithms for DNA histogram analysis.