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 FacsCelesta LSRII and FacsCaliburare 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 8:30 AM on the LSRII should turn the machine on early, 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 a Saphhire 4884 laser, a Coherent Cube 640 laser and a Coherent Cube 4057 laser and is capable of detecting 13 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 GFP, the most commonly used fluorochromes are FITC, PE, APC, PI, PE-TXRD, PE-CY5, and APC-CY7. The MoFlo is also equipped with an Aerosol Evacuation Unit6 which will guarantee that no aerosols or particulates will escape from the sorting chamber. This will allow for safe sorting of human samples.
A BD Biosciences FACSAria IIIu2,5 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, 640 nm, 561 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 and PerCP-Cy5.5. The 561 nm laser can excite dyes like PE, PI, and PE-Cy5, PE-Cy5.5 and PE-Cy7. The 633 nm laser is used to detect APC, APC-Cy7 and APC-Cy5.5. 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 Quantum Dots, 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 BD LSRII3 analyzer is equipped with 405 nm, 488 nm, 561 nm and 640 nm lasers. This digital instrument is a good companion to the FACSAria and is capable of detecting up to 19 parameters. Three fluorescent parameters from the 488 nm, three from the 640 nm, five from the 561 nm and six from the 405 nm.
The BD FacsCelesta8 analyzer is equipped with a 405 nm, 488 nm and 640 nm lasers and can detect 14 parameters. This digital instrument is capable of detecting 4 fluorescent parameters from the 488 nm laser, 5 parameters from the 405 nm laser and 3 parameters from the 640 nm laser.
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 dongles for Diva and Cellquest Pro that can be loaned out 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 own 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 to 17 fluorescent parameters.
4The MoFlo replaced the Enterprise II laser with a solid state Sapphire 488 laser with funds from an OCASCR grant in 2014.
5The FacsAriaIIu was upgraded to the FacsAriaIIIu with the addition of a Cube 561nm laser from funds from an OCASCR grant in 2015.
6The MoFlo was upgraded with an Aerosol Evacuation Unit with funds from an OCASCR grant in 2015.
7The MoFlo was upgraded with the Coherent Cube 405 laser with supplemental funds from an OCASCR grant in 2016.
8The FacsCelesta was purchased from funds from COBRE funds in 2016.
All current and future users must submit the preapproval form before arriving for their sort. This approval form will only need to be submitted once per experiment design but must be resubmitted before any new experiments or if the experiment protocol is altered in any way. Please email the approval form to: Jacob-Bass@omrf.org.