This proposal seeks funding to replace the flagship instrument in the University of Pittsburgh Cancer Institute (UPCI) Cytometry Facility (CF), our biocontained High Speed Sorter. The existing instrument, a MoFlo in which the fluidics components are contained within a certified Class I Safety Cabinet, was custom assembled 12 years ago through a Shared Instrumentation Award and $110,000 additional funding from the UPCI. Over its lifetime it has serviced literally hundreds of NIH-funded investigators, including many who make use of the ability to sort live human cells while exercising Universal Precautions. Because of our continuous efforts to update the MoFlo (adding lasers, PMT channels, 4-way and plate sorting, chilled sample station) this instrument has provided excellent service well beyond its expected service life. Despite CF Supervisor E. Michael Meyers' training as a MoFlo field service engineer, the MoFlo has been increasingly out of service (at the time of this writing it has been nonfunctional for 3 weeks due to failure of the obsolete laser water chiller/heat exchanger; the pump is being remanufactured). This places an unacceptable hardship on Facility Users, many of whom require our unique biocontainment capabilities, our expertise in multi-parameter multi-way sorting, and continuity in technique and instrumentation. The newly released Beckman-Coulter Astrios retains the functionality of our current instrument, replacing: 1) The class I biosafety cabinet with a well integrated class II cabinet;2) Obsolete analog electronics with digital electronics;3) Obsolete photomultipliers (PMTs) with higher sensitivity PMTs;4) Energy inefficient water-cooled lasers with diode lasers, reducing energy consumption by 95%. The proposed Astrios will also provide a new laser line (561 nm) and increase the maximum number of fluorescence channels from 8 to 14. Through our own independent testing of the Astrios with beads and live cells, we have documented a significant increase in sensitivity, resolution, sort speed and recovery over our existing MoFlo. The requested enhancements will allow us to continue to provide the best possible cell sorting resource to our large group of NIH-funded investigators.
