Cytometry Core
The goals of the Cytometry Core of the Nathan Shock Center of Excellence in the Basic Biology of Aging are to:
- To make readily available flow cytometry, cell sorting, confocal image cytometry, and laser capture microdissection instrumentation and techniques to research projects in the basic biology of aging.
- To assist investigators in finding appropriate ways to apply these techniques to their research objectives.
- To assist investigators in the application of computer software for flow and image cytometry data analysis and presentation, including customized software programming, as needed.
The Nathan Shock Center Cytometry Core has specialized in providing assays that are less commonly available at other facilities, require special expertise to perform and interpret, or both. In addition, this core provides access to confocal image cytometry and laser capture microdissection. The range of applications for confocal analysis extends for localization of transgenic gene products to measurement of telomere length. Finally, both laser capture microdissection and flow cytometry play important roles in purification of cell subsets for subsequent molecular analyses, including microarrays and proteomics.
The Cytometry Core utilizes a state-of-the-art high speed (>108 cells/hr) cell sorter, the Influx, from Cytopeia (Seattle, WA). This instrument was designed by Dr. Ger van den Engh, as an update to his previous design of the Cytomation MoFlo. It is a 3-laser, 12-parameter instrument, with full integration of computer control and the highest speed sorting capacity of any commercial instrument. A PALM laser catapult microdissection instrument is also available for shared use.
A few of the special assays that the Cytometry Core has continued to apply to aging-related research are:- Multiparameter cytometric assays to monitor physiological changes during dell injury, apoptosis and necrosis. Multiparameter assays of physiological changes in cells have been a past focal point of the Cytometry Core. The measurement of mitochondrial membrane potential has become important in assessing the opening of mitochondrial transition pores, an early and important event in apoptosis. The mitochondrial membrane sensitive dye CMXRosamine has been used in conjunction with assays of glutathione, NADPH, cytosolic oxidant levels, the level of mitochondrial membrane lipid cardiolipin (by nonyl acridine orange fluorescence) and cell cycle parameters to quantitate and delineate events occurring during apoptosis. In these assays cells exhibiting concordant or discordant changes in fluorescence intensities are classified with regards to several metabolic parameters. For instance, changes in mitochondrial NADH level can be determined simultaneously with the mitochondrial membrane potential. Thus, it can be determined that NADH and the mitochondrial membrane potential undergo a concordant or discordant change during aging of cells. Such a concordant or discordant change may reflect a certain pathway of apoptosis.
- Cytometric assay of proliferative survival. This assay has been developed to provide a rapid and sensitive alternative to classical clonogenic survival assays. It has a great advantage in cases where cells clone poorly (e.g. Werner Syndrome fibroblasts), are technically difficult to clone (e.g., lymphoblastoid cells), or when only few cells are available for the assay. The assay uses BrdU incorporation as an indicator of proliferation, and calibrates surviving cell numbers using an internal particle control. We have also shown that this assay can be performed simultaneously with immunofluorescence staining to identify cell subsets. A principal application has been to the study of drug sensitivity of Werner Syndrome cells.
- Quantitation of telomere length
- Telomere measurements by confocal microscopy. Fluorescence in situ hybridization (FISH) with a PNA telomere probe provides an accurate and specific indication of telomere length. This has conventionally been done in cultured mitotic or interphase cells. We have shown that this method can be applied to tissue sections, and that this is especially useful when the telomere lengths of cellular subsets within tissues is to be determined. In the chronic inflammatory disease ulcerative colitis, we have used this method to show that increased chromosomal instability is related to telomere attrition. We have taken advantage of the statistical and programming experience of the Cytometry Core to compare and validate a number of methods and algorithms for analyzing the confocal images and most accurately and reproducibly deriving relative telomere lengths. The results of this study have been published (O'Sullivan JN, Finley JC, Risques R, Shen W-T, Gollahon KA, Moskovitz AH, Gryaznov S, Harley CB, Rabinovitch PS. Telomere Length Assessment in Tissue Sections by Quantitative FISH: Image Analysis Algorithms. Cytometry, Apr; 58A(2):120-31, 2004).
- Telomere Measurements by flow cytometry. Peter Lansdorp (Fox Chase, Vancouver BC) has previously shown that the intensity of Telomere PNA FISH can be quantitated in leukocyte subsets by flow cytometry. For Center investigators who are interested in telomere maintenance and telomere attrition, it would be very powerful to be able to measure the rate of telomere length reduction per cell division. Since cells in culture grow at heterogeneous rates, this can only be done presently by obtaining a population average by serial measurements. We have assisted Dr. Mark Wener in the recent development of a novel assay that measures the rate of telomere attrition per generation in a single flow assay. The cells are initially labeled with a membrane permanent dye, CFSE, that is cleaved by esterases to form a stably retained fluorescent compound; at each cell division the intensity of stain is halved, By discriminating cells with full, half, quarter, etc intensity after a period of culture, one can detect cells that have divided 0 through 9 times.
- Telomere measurements by real-time Q-PCR. Because of the great interest in telomere biology in aging, the Cytometry Core rounds out its support of this field by offering assistance with real time qPCR measurement of telomere length, using the method originally described by Cawthon (Cawthon RM. Telomere measurement by quantitative PCR. Nucleic Acids Res. 2002 May 15;30(10):e47). In our hands this method offers excellent accuracy and reproducibility (inter-experimental CV<7%), using very small amounts of nuclear DNA.
- Sorting with preservation of RNA for microarray studies. Flow cytometry can be a rapid and efficient method of purifying cell subsets for molecular analyses, including gene expression and proteomics, particularly in cases where admixtures of cells may yield insensitive, confusing or misleading answers. The high-speed sorter can process in excess of 108 cells/hr, providing adequate numbers of molecular assays even when the desired cells are relatively rare. In particular, we have shown that epithelial cells can be labeled with anti-cytokeratin antibodies after fixation with RNAlater and that high quality RNA can be recovered after sorting, even when no special treatment (elimination of RNAases) of the cell sorter is used.
- Assays of DNA damage by alkaline unwinding. A flow cytometric assay of DNA damage based on alkaline unwinding has been developed and shown to have excellent sensitivity and reproducibility. This assay has been applied to aging research to examine DNA damage in response to oxidative challenge in cells from control and transgenic mice and cells from Werner Syndrome.