Shared Resource: Cell Sorting Facility

点击翻译按钮获取中文摘要

CELLULAR/MOLECULAR ANALYSES SHARED RESOURCE: CELL SORTING FACILITY (CSF) PROJECT SUMMARY/ABSTRACT The Cell Sorting Facility (CSF) provides Cancer Center members access to and technical support for the technology of flow cytometry, a method for analyzing and isolating (sorting) cells in a complex mixture, based on a variety of parameters, including light scatter and fluorescence. The CSF under the direction of Richard Hardy, PhD (BCDF) and a staff of 1.3 FTEs has a goal to provide the highest quality analytic and sorting capabilities to Cancer Center members. As flow cytometry technology has matured, it has become an essential component of research, utilized in a diverse array of laboratories. 31 Cancer Center members used the CSF in calendar year 2014. 94% of service hours logged in the CSF during 2014 were in support of peer-review funded research. 48 Cancer Center members from all 5 Research Programs used the CSF in the period 2011- 2014. Due to the complexity, cost and caliber of the instrumentation, the services are best facilitated though the centralized CSF. It would be cost prohibitive to have individual flow cytometers available in each laboratory. The nature of flow cytometry analysis and/or sorting is not really appropriate for an external (commercial) service. That is, samples are often live cells, prepared shortly before analysis (therefore quite perishable) and many experiments involve the use of diverse combinations of novel reagents (fluorochrome-labeled monoclonal antibodies). The 28 years of experience and expertise of Hardy is key in implementing state-of- the-art technology and innovative approaches. Hardy's leadership coupled with institutional commitment led to the purchase of a highly customized FACSAria ($509,500). This new instrument optimally excites PE and PE tandem conjugates with a green laser, providing greater signal-to-noise relative to blue 488nm excitation. The green laser also enables detection of certain fluorescent proteins that could not be detected using the previous Vantage sorter. The Aria also runs the current version of instrument control software with a programmed auto- calibration at setup each morning. The LSR-II was also upgraded to enhance its capabilities, by adding a green laser (enabling most efficient excitation of PE and PE tandems), replacing the red laser to increase laser power, and increasing the number of fluorescence detectors. This instrument also runs the current instrument control software with auto-calibration each morning. This auto-calibration software on the Aria and LSR tracks the sensitivity in each channel and so provides added quality assurance, flagging potential problems before they progress to failure. The in-house developed data management system, Flow-LIMS, that allows all Cancer Center members to annotate, access, and manage flow cytometry data on computers in their local laboratories, has been designed to facilitate sharing of experimental data, and it helps to foster collaboration between laboratories. The CSF is advised by a dedicated Facility Advisory Committee (FAC) that meets annually. FAC recommendations are directed to a governing Facility Parent Oversight Committee, that serves to ensure that the CSF continues to have the necesssary resources to provide the highest quality services.