Flow Cytometry Laboratory Facilities

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 information that the Flow cytometry 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.

Equipment

Main Science Laboratory:

We maintain a quality assurance program for all Flow Cytometers using guidelines from various regulatory agencies.  These include the New York State Department of Health, the College of American Pathologists, the Joint Commission on Accreditation of Healthcare Organization and the Clinical Laboratory Improvement Act.  Even through only two instruments, a FACSCalibur and the FACSCanto, are used for clinical reference services, we apply the exact same quality assurance and control procedures to each of our instruments.  The following equipment is located in the Science building main laboratory.

A Shared Instrumentation Grant submitted in March 2002 and funded in January 2003 was used by the facility to acquire a FACSAria which has sorting capabilities, nine fluorescent parameter plus side and forward scatter. The instrument utilizes separate interrogations for each laser intercept, ensuring proper excitation/ detection of tandem dyes. All BDB cytometer instruments are designed with individual excitation laser intercepts within the fluid stream for proper tandem dye detection. The FACSAria is operated by Earl Timm who assists investigators needing sorted cells.

The FACSDiVa (Vantage) was developed at RPCI in collaboration with Becton Dickinson Biosciences (BDB). It is capable of ten parameter data acquisition with eight colors of fluorescence detection using separate excitation wavelengths from three lasers including UV (488nm, 633nm, 350nm). It has bio-containment capabilities, so that infectious specimens can be safely sorted and a single cell deposition system used to sort cells into microtiter plates. The FACSVantage was upgraded in 2004 with the FACSDiVa option converting it from analog to digital electronics and enhancing the software user interface.

In October 2006, a BDB LSRII was purchased though Institution funds for the Resource. The LSRII is equipped with three lasers (405 nm, 488 nm, and 633 nm) and has six fluorescent detectors plus scatter off the 488 nm laser line, three fluorescent detectors off the 405 and three fluorescent detectors off the 633 laser line, fourteen parameters total. This instrument is available to all users and addresses the increasing demand for multiparameter analysis by cancer center members. The LSRII is available 24 hours a day, seven days per week to all qualified investigators.

The Resource also has two BDB FACScans (488 nm laser, three fluorescent color detectors, 5 parameters), two FACSCaliburs (488 laser and 635 nm diode, 4 fluorescent color detectors, 6 parameters), a BDB LSRI and cytometer brass board which are all operational. One FACScan and both FACSCaliburs are available 24 hours a day, seven days per week to all qualified investigators. The other FACScan, BDB LSRI and brass board cytometer are used as breadboard cytometers, which can be configured with other lasers or detectors depending on the needs of the investigator. Currently, one of the FACScans is equipped with a 785 nm laser and an avalanche photo diode (APD) for work in the near IR (emission greater 820 nm) and the LSRI is configured with four lasers, 405 nm, 488 nm 525 nm and 785 nm and has eight fluorescent detectors (7 PMTs and 1 APD). The brass board cytometer has 16 parameter detection capabilities for experiments utilizing multi-element APD detection arrays.

The Research core also has access to a third FACSCalibur, and a BDB FACSCanto. The FACSCanto was acquired in 2004, as part of a reagent rental package and is a six-color, eight-parameter, two-laser (488nm and 633nm) instrument with automatic sample loading. Both of these instruments are reserved during the day for clinical use, when they are used in part to acquire data on samples received for clinical research protocols. These instruments are available in the afternoon and evenings to investigators on an as needed basis.

BDB FACS Canto II??

The facility also posses the ability to profile optical filters used by our flow cytometers as an addition to quality control and aid in researcher experiment design. A new Jasco V530 spectrometer was modified to measure the spectral response of filters and dichroics from 190nm to 1100nm using interactive analysis software. A custom built filter interrogation holder was constructed by our facility which includes a rosette for precise angles settings while profiling dichoic mirrors. Optic spectral response for our instruments is listed on this WEB site.

In March 2000, RPCI purchased a Luminex-100 to upgrade the Resource’s ability to meet the demand for multiplexed, soluble bead array (SBA) immunoassays of cytokines, chemokines and growth factors. Because of the increasing number of assays and the precision of pipetting required for the SBA, RPCI purchased a Tecan liquid handling robot in 2004. Funding for the Tecan was provided by a combination of Institute support and increased chargeback revenues.

Satellite Laboratory

The following equipment is located in the BLSC satellite laboratory. In 2007, a Beckman Coulter FC500 was acquired for the BLSC satellite as part of a reagent rental package. The FC500 has a 488 nm laser and coaxial with a 635 nm red diode for excitation. Care must be taken in selecting of detection dyes due the coaxial nature of the two laser beams. This is a five-color, seven-parameter instrument with optional automatic sample loading. The FC500 was installed and functional in March 2007. It is available 24 hours a day, seven days per week to qualified investigators.

In 2006, a Shared Instrumentation Grant was funded to acquire an Amnis ImageStream 100, which is designed to acquire six multispectral images of individual cells at rates of 5,000 cells per minute. The ImageStream combines the power of image-based microscopy with analysis of statistically robust cell numbers obtained by flow cytometry. The instrument at RPCI is one of only 20 of these instruments installed world-wide and became functional in February 2007. Latest options installed are a second laser,405nm, and extended depth of field.

Hitachi H-600 electron microscope equipped with an electron energy filter which permits electron spectroscopy for elemental analysis and clarification of images blurred by inelastic electron scattering.

ETEC Autoscan scanning electron microscope equipped with a backscattering electron detector.

Leica TCS SP2 Spectral confocal fluorescence imaging system with 4 available laser excitation sources (405 nm diode laser, an argon laser with lines at 457, 477,488 and 514 nm, and two HeNe lasers, one at 543 and one at 633 nm). It has three continuously adjustable spectrophotometer detectors. Standard analysis and deconvolution software allows 3-dimensional reconstruction of the obtained images

New equipment purchased for the Cell Analysis Facility includes two state-of-the-art Leica Live Cell Imaging Systems. The Leica AS MDW DMI6000 was delivered in June 2006 and the Leica AF6000 LX was installed in March 2007. These instruments were purchased with Institute funds and can acquire standard or confocal images in temperature-, humidity and CO2-controlled environments. The automated image capturing with user-defined time steps and observation duration allows for time kinetic studies of intracellular distribution of fluorescent probes.

Software

A variety of software programs are available primarily because of the facility’s interest in new approaches and the concomitant beta testing that is carried out by staff. The Flow Cytometry Resource is thus kept up-to-date with the latest in software development. All of the programs listed below are available for use by staff and investigators, free of charge, in both the Main and Satellite Computer Laboratories.

  • CELLQUEST is the data acquisition and analysis program from BDB and operates in the Macintosh environment on the FACScan and FACSCalibur cytometers.
  • FACSDiVa is BDB's next generation data acquisition and analysis program and operates in the PC Windows environment on the FACSAria, FACSCanto, FACSCanto II and Vantage.
  • WinList is a multiparameter data analysis program from Verity Software House. Since most users have PCs, this is by far the most popular program. RPCI has a site license for this program making it available to all Roswell Park employees at 85% off list price for use in the lab or at home. There are a limited number of network accessible licenses through the CITRIX server for Roswell authenticated users to analyze data from their own computer but do not have the software actually installed.
  • ModFit, also from Verity, is designed to analyze DNA histograms and has advanced capabilities for aiding the user in handling debris and “sliced” nuclei often found in preparations from solid tissues. The program automatically links to WinList, so that combining immunophenotyping with DNA content analysis can take place simultaneously.
  • FCSExpress is a multiparameter analysis program developed by DeNovo Software that is similar to WinList in capabilities. It has the ability to easily print/save layouts, save statistics to Excel and create presentation quality output straight into PowerPoint. Multiple FCSExpress licenses were purchased by the Resource and have been placed on a server making this program available to anyone with an Institute account.
  • IDEAS Software is the data analysis software used to view, compensate, and analyze ImageStream data files. It calculates 35 morphological and signal intensity features on each of the up to 6 images collected per cell. For each experiment, a template is created which then can be easily applied to similar experimental designs.
The Flow and Image Cytometry Resource:

The Flow and Image Cytometry Resource provides for the complete flow cytometric and image analysis needs of RPCI’s clinical and research staff. This includes: (i) sample staining and preparation, (ii) flow cytometric and image data acquisition, (iii) confocal microscopy, (iv) live cell imaging, (v) transmission and scanning electron microscopy, (vi) data analysis, (vii) sorting and (viii) Luminex multi-analyte bead array assays.

Flow Cytometry Services:

This resource provides a comprehensive set of flow cytometric services to our users. The facility is equipped with 5 data acquisition cytometers that are available to trained users 24 hours a day, 7 days a week. These range from a 3 color FACScan to a 13 color LSRII. Data can be analyzed on site or at the investigators desk using a server based analysis software. We maintain two sorters with fulltime operators to assist researchers with their experiments. Investigators may also bring their unprocessed samples to our staff, who will prepare and acquire it on a cytometer, analyze it, and help them interpret the results. The Resource employs a fulltime engineer who routinely performs preventative maintenance maintaining the instrumentation at or exceeding factory recommendations. Investigators with unusual flow cytometric applications (such as fluorochromes requiring atypical excitation or emission wavelengths) may take advantage of the engineer’s expertise to design and build a breadboard cytometer to meet their requirements.

Image Stream:

In March 2007, the Resource obtained an Amnis ImageStream flow cytometer through a shared instrument grant at a cost of approximately $200,000. This instrument combines the efficiency and quantitative nature of flow cytometry with the visual power of microscopy. Our experience with this instrument is new, and staff is currently assisting the 9 active investigators with all aspects of data acquisition and analysis as we familiarize ourselves with the technology. Data analysis is complicated by the availability of 41 parameters per image, which are calculated for all six images plus multiple user defined parameters. Once an analysis template for a particular application is created, however, the analysis is fairly straightforward. Most users will require assistance from the Resource staff developing templates, but once these are created will be able to analyze their own data on site. Dr. Minderman, in a consortium agreement with the Amnis Corp, was awarded an R21/R33 grant by the NIH in April 2007 to evaluate the clinical application of this technology. This close collaboration with the Amnis will assure that our facility will be on the forefront of any new hardware and software developments related to this technology.

Confocal Microscopy:

Confocal microscopy is performed on tissue sections and cells stained with various fluorescent markers. A Leica TCS SP2 Spectral Confocal Microscope, purchased with Institute funds, with a wide fluorochrome application range including capabilities for viewing Hoechst and DAPI nuclear stains (blue diode), purchased with RPCI funds, forms the foundation of our confocal microscopy services. A dedicated full time technologist is available by appointment to assist users with the confocal microscope. Individualized training is also available for users desiring to perform their own data acquisition.

Leica Live Cell Imaging Systems:

This Resource has two state-of-the-art Live Cell Imaging Systems (Leica AS MDW DMI6000 and Leica AF6000LX) that were delivered in June 2006 and March 2007, respectively. These instruments were purchased by Institute funds. The live cell imaging systems enable time kinetic studies of intracellular fluorescent targets in a temperature-, humidity- and CO2 -controlled environment. Time intervals, observation duration, number of positions observed per time point and number of Z-positions acquired per field of observation are fully operator controlled. Use of the live cell imaging systems is available by appointment and trained facility personnel are available to the investigators to assist in experimental and instrument set-up and data analysis.

Electron Microscopy Services:

Electron microscopy services include transmission (Hitachi H-600 and scanning EM (Etech Autoscan)) purchased with Institute funds. In addition to routine tissue processing (including special processing of primary cultures, cell monolayers, and subcellular organelles), several specialized techniques are offered, including pre- and post-embedding immunocytochemistry, scanning immuno-EM, quantitative EM autoradiography, cryoultramicrotomy, negative staining, critical point drying, extreme angle rotary platinum shadowing, and Kleinschmidt technique. A technician, trained and experienced in all aspects of sample preparation, data acquisition and photographic documentation related to these techniques, is available to investigators by appointment.

Soluble Bead Array:

Soluble Bead Array is a major focus of Research and Development within the Flow Cytometry Laboratory over the past decade has been the development of an operational capability to measure cytokines using the soluble bead array (SBA) technology based on the Luminex-100 Cytometer. The SBA Laboratory has focused on cytokine, chemokine and growth factor assays. Currently we offer 67 different human, 28 mouse, and 12 rat analytes (see http://www.rpciflow.org/assays.html for specifics). The Resource has the in-house expertise and capacity to add additional analytes for investigators providing a suitable antibody pair is available.