Flow Cytometry

About

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 and S402.

Jacob Bass and Diana Hamilton, Ph.D., 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 Celesta, LSRII and Aurora.

Scheduling

The Flow Cytometry Core schedules equipment through an iLab calendar. Your PI needs to have registered with iLab before lab members are able to schedule an appointment.

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 Aurora are cell analyzers and can be reserved 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 at least 10 minutes for warmup. The Aurora should be allowed to warm-up for at least 30 minutes. Users can edit and delete their times for the analyzer calendars.

There is a kiosk workstation for starting and stopping your time. The billable time will be based on the kiosk.

iLab calendar
https://OMRF.ilab.agilent.com/service_center/show_external/4780

Refer to these documents for help getting started
How to Use Kiosk
New User Registration
PI Registration

Fees

Standard Charges for Core Facility

Flow cytometry analysis –Aurora, FacsCelesta & LSRII
Operator assisted:  $40/hour
Independent user:  $20/hour
Minimum charge 15 minutes

Cell sorting – MoFlo, FACSAria
Set-up fee:  $13
Hourly rate:  $92/hour
Analysis:  $40/hour
Minimum charge 1 hour

Corporate Users
Flow Cytometry Analysis: $50/hr
Flow Cyometry Sorting: $200/hr and Set-up fee: $25

Helpful Links

Fluorofinder Panel Builder
https://app.fluorofinder.com/omrf

Cytek Aurora Resources
https://cytekbio.com/blogs/resources?

“What is Cytometry” and “Learning Diva Software”
http://www.bdbiosciences.com/us/s/training/e-learning

Fluorescence Spectrum Viewers
BD Spectra Viewer
BioLegend Spectra Viewer
ThermoFisher Spectra Viewer
http://www.cyto.purdue.edu/
http://facs.scripps.edu/
http://www.dako.com/
http://www.bdbiosciences.com/

Fluorochrome information
http://www.zeiss.com/C12567BE0045ACF1/Contents-Frame/CF19CAEC78522802C1256AB0005242E9
http://flowcyt.salk.edu/fluo.html
http://en.wikipedia.org/wiki/Fluorophore

Instrumentation

The 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 20,000 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, APC-CY7 and Brillant Violet dyes. 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 IIIu^2,5 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.

The Cytek Aurora^9,10 is a full spectra analysis flow cytometer. It has five lasers: 355nm, 405nm, 488nm, 561nm and 640nm. It can detect three scatter channels and 64 fluorescent channels. The Aurora can also measure the volume of acquired samples.

To assist researchers with analysis, the Flow Lab has a MacMini with SPICE and FlowJo and a PC with Diva, and FlowJo. Users can also inquire about signing up for OMRF’s FlowJo site license. We provide a transfer file on the network and have CDs and DVDs available to make copies of data files. We make routine backups of the data, but all users should frequently back-up their own data as well.

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.
⁹The first Aurora was purchased with an S10 grant from NIH in 2020.
¹⁰The second Aurora was purchased with grants from OCASCR and PHF in 2022.

Procedures

All users must read through the appropriate SOP for the instrument that they are using and abide by the safety protocols at all times.

SOP for the FacsAria II
SOP for the Aurora
SOP for the FacsCelesta
SOP for the LSRII
SOP for the MoFlo XDP

Contact