The Flow Cytometry and Cell Sorting Core Facility supports many OMRF investigators by providing training, analysis and multi-parameter, high speed cell sorting. The facility is housed in Room E402.
Mr. Jacob Bass and Dr. Diana Hamilton have completed the factory courses for these instruments and conduct all cell sorts for users. They also perform routine maintenance and data base archiving on the instruments and provide guidance on staining, sorting, special applications and analysis. In addition, they train postdoctoral fellows, graduate students and technicians in operation of the FACSCalibur and LSR II.
User name is flowlab and the password is cytometry. The FacsAria and MoFlo are cell sorters and can be scheduled two weeks in advance. Appointments can be made from the times of 10:00 am to 5:00 pm with an hour off for lunch. Users cannot edit or delete their times for the sorters and should contact the lab to make changes. Cancellations for the Aria and MoFlo should be made 24 hours in advance; otherwise, there is a 1 hour cancellation charge.
The FacsCalibur and LSRII are cell analyzers and can be scheduled one month in advance. Trained users who feel confident to use our analyzers after hours (after 5 PM on the weekdays and the whole weekend) may do so. Any user that signs up before 9:30 AM on the LSRII should turn the machine on, since it requires a longer warm up period. Users can edit and delete their times for the analyzer calendars.
Our Beckman Coulter Moflo XDP1 sorter purchased in 1998 is equipped with an Enterprise II 621 laser, a Coherent Cube 640 laser and is capable of detecting 9 parameters. It is routinely used for 2-way and 4-way sorts at speeds of up to 30,000 cells/second as well as single cell deposition sorts. In addition to green fluorescent protein, the most commonly used fluorochromes are FITC, PE, PI, PE-CY5, PerCP-Cy5.5, PE-Cy7, APC, Alexa700 and APC-CY7.
A BD Biosciences FACSAria IIu2,4 desktop cell sorter is also available for cell sorting. This instrument has no counterparts with respect to capabilities and ease of use. It utilizes 488 nm, 633 nm and 407 nm air-cooled lasers with advanced optics to detect up to 18 independent signals. The 488 nm laser can be used to detect common fluorochromes including GFP, FITC, PE, PI, and PE-Cy5, PerCP-Cy5.5, PE-Cy7. The 633 nm laser is used to detect APC, APC-Cy7, Alexa700. These fluorochromes are most commonly used by our investigators who sort hematopoietic progenitors or B cell subsets from tonsils. Finally, the 407 nm laser is appropriate for FRET experiments, exciting Brilliant Violet Dyes and for using Hoechst 33342 to study Side Population. The FACSAria also has an Aerosol Containment Device to ensure that any aerosolized particles are contained properly and making it safer to run human tissue samples. The LSRII3,4 analyzer is equipped with 405 nm, 488 nm, 561 nm and 633 nm lasers. This digital instrument is a good companion to the FACSAria and is capable of detecting up to 19 parameters.
A BD Biosciences FACSCalibur cytometer is capable of four color fluorescence analysis. Utilizing a 488nm laser exciting up to three fluorescent parameters and a 633nm laser exciting one fluorescent parameter.
To assist researchers with analysis, the Flow Lab has a MacMini with Cellquest Pro and FlowJo and a PC with Diva and Summit. Users can also inquire about signing up for OMRF’s FlowJo site license. We also have three dongles for Diva, FlowJo and Cellquest Pro that can be loaned to users. We also provide a transfer file on the network and have CDs and DVDs available to make copies of data files. Although we make backups of the computers, all users should frequently back-up their data.
1The MoFlo was upgraded to a MoFlo XDP in 2012 from funds from an OCASCR grant. New PMTs, digital processors, computers and a solid state 640nm (Red) laser were installed, allowing better data resolution and performance. 2The Aria I was upgraded to an Aria IIu in 2012 from funds from an OCASCR grant. Fluidics were upgraded to a more stable and functional system, allowing for more reliable day to day performance. 3The LSR II was upgraded to a 4 laser system in 2010 from funds from an OCASCR grant. New 405nm (Violet) and 561nm (Green) lasers were installed and the 640nm (Red) laser was replaced. We expanded our capabilities from 12 parameters to 16 parameters. 4The FacsAria and LSRII were upgraded with three additional PMTs to the Violet laser from funds from an OCASCR grant.