The shared resource provides for the complete flow cytometric and image analysis needs of RPCI's clinical and research staff, including (i) sample staining and preparation, (ii) flow cytometric and image data acquisition, (iii) confocal microscopy, (iv) live cell imaging, (v) ImageStream cytometry, (vi) data analysis, (vii) sorting, and (viii) Luminex multi-analyte bead array assays. The shared reource staff are available to assist investigators with the design and development of their experiments.
The shared resource provides a comprehensive set of flow cytometric services. The resource is equipped with five data acquisition cytometers that are available to trained users 24 hours per day, seven days per week. These range from a 4-color FACSCalibur to an 18-color LSRII. Data can be analyzed on site in the facility or at the investigator's desk using server based analysis software. Two sorters with full-time operators assist researchers with their experiments. Investigators may also bring their unprocessed samples to the staff, who prepare and acquire it on a cytometer, analyze it, and help them interpret the results. The resource employs a full-time engineer who routinely performs preventive maintenance that often exceeds 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.
The ImageStream platform combines the efficiency and quantitative nature of flow cytometry with the visual power of microscopy. Dr. Hans 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 relatively new technology. Through the close collaboration with the Amnis Corp., the facility was invited to be a beta-test site for the ImageStream technology and has since beta-tested two new generations of this platform (the ImageStream-X and the ImageStream-MKII). The continuing collaboration with Amnis Corp (now part of Merck-Millipore) assures that the facility is on the forefront of any new hardware and software developments in this area.
Confocal Microscopy is performed on tissue sections and cells stained with various fluorescent markers. RPCI funds were used to purchase a Leica TCS SP2 Spectral Confocal Microscope with a wide fluorochrome application range, including capabilities for viewing Hoechst and DAPI nuclear stains (blue diode), which forms the foundation of our confocal microscopy services. A dedicated full-time technologist is available by appointment to assist users. Individualized training is available for users desiring to perform their data acquisition.
Leica Live Cell Imaging Systems
The Leica AF6000LX live cell imager was purchased with Institute funds in March 2007. The Live Cell Imaging System enables 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 System is available by appointment, and Resource personnel are available to assist investigators with experimental and instrument setup and data analysis.
Soluble Bead Array
A major focus of research and development within the resource 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 and has recently validated and implemented the magnetic bead array platform which has significantly improved the efficiency of the assay. The Resource has the in-house expertise to assist investigators in defining multiplex kits for human, mouse, and rat analytes from commercial sources and has a dedicated operator to assist the investigators with data acquisition and analysis.
Training and Education
A combination of lectures, hands on wet labs and individualized instruction are used to train resource users. Users new to flow cytometry are required to attend an orientation class designed to introduce and demonstrate instrument standard operating procedures. Students learn basic flow cytometry theory, proper instrument startup, setup (experimental design and controls are emphasized) and shutdown procedures. Optional courses are taught to address topics such as: (i) fluorochromes & compensation, (ii) immunophenotyping of hematopoietic and cultured cells, (iii) DNA cell cycle staining and analysis, and (iv) methods of measuring apoptosis. Instruction also is provided in the use of the confocal fluorescence imaging system as well as introductory and advanced courses for ImageStream applications and data analysis. In 2012, a total of 12 courses were attended by 84 students. This approach has been very successful, since researchers get first-hand insight into the capabilities of the resource, and are better able to design more sophisticated experiments and properly interpret the corresponding results.