Flow Cytometry Services and Instrumentation

Our Flow and Image Cytometry Shared Resource (FICSR) facility houses five Becton Dickinson flow cytometry analyzers (LSRIIA, LSRIIB, Fortessa A, Fortessa B and Fortessa C), a Zetaview small particle analyzer and a flow cytometry sorter. These instruments are accessible 24/7 to all trained investigators. In addition, there are several breadboard custom instruments, a Beckman Coulter Gallios, and two flow cytometry sorters that are available for use by appointment with our operators during regular business hours (Monday – Friday, 8am – 6pm).

During weekdays, our staff performs start-up procedures and daily QC on the analyzers. During weekends and on holidays, users are responsible for these procedures. Investigators are expected to work in the facility independently, but FICSR staff is available during regular business hours for consultation. Our facility, including the user-accessible instrument room, is designated a BSL2+ area which means that applicable biosafety precautions should be adhered to  including wearing personal protection devices  such as lab coats and gloves.

Users can reserve time on the equipment and monitor availability of the equipment using the online FOM calendar. Individual access to this system is granted only after completion of the free Introduction to Flow Cytometry class (sign up here). The class outlines the guidelines and requirements for use of the facility, and gives a basic overview of the technology. Further one-on-one training to become proficient on the instruments is available by appointment with our staff.

Instrument use is charged by actual usage time, or 80% of calendar reservation time, whichever is greater. Reservations can be altered up to two hours before the start time. Reservations that do not begin within 30 minutes of the reservation start time will be released and charged at 80% of reserved time. A reduced rate is offered for evening or overnight hours (8pm – 8am), weekends and holidays.

Flow Cytometers

Custom Flow Cytometers

The Flow Cytometry Electronics Shop utilizes an optical bread board, supporting digital acquisition electronics, fluidics and cuvette flow cell for custom flow cytometric experiments. Custom technology available for studies include contiguous multispectral detection using diffraction grating optics, Infra-red flow cytometry, fluorescence detection beyond 1100 nm and an optical breadboard flow cytometer open to diverse configurations. There are 20 detection parameters each with 262,144 channel (18 bit) resolution and 5-1/2 decade dynamic range electronic processing. Various laser lines also available for excitation.

Researchers interested in using this technology should contact Ed Podniesinski (716) 845-8775.

Flow Cytometry Analyzers

Technical Specifications

  • 5 laser, separate interrogation of sample for each laser
  • 18 color detection + 2 scatter parameters (from 488nm)
  • Hydrodynamically focused sample interrogated within a quartz cuvette flow cell
  • Sample aspiration rates at 12ul, 35ul, 60ul per minute
  • 262,144 channel (18 bit) dynamic range on all parameters
  • PC Windows 7, Diva version 8.0.1

Excitation Sources

  • 355nm 100mw, JDS Uniphase Laser system
  • 405nm 25mw, Coherent Diode laser system
  • 488nm 20mw primary laser, Coherent Solid State Laser system (Sapphire)
  • 561nm 50mw, Coherent Solid State Laser system
  • 640nm 40mw, Coherent "Cube" Solid State Laser system

Emission Detected

Click image to enlarge

Technical Specifications

  • 4 laser, separate interrogation of sample for each laser
  • 16 color detection + 2 scatter parameters (from 488nm)
  • Hydrodynamically focused sample interrogated within a quartz cuvette flow cell
  • Sample aspiration rates at 12ul, 35ul, 60ul per minute
  • 262,144 channel (18 bit) dynamic range on all parameters
  • PC Windows 7, Diva version 8.0.1

Excitation Sources

  • 488nm 20mw, Coherent Solid State Laser system (Sapphire)
  • 640nm 40mw, Coherent "Cube" Solid State Laser system
  • 405nm 100mw, Coherent "Cube" Solid State Laser system
  • 561nm 50mw, Coherent Solid State Laser system

Emission Detected

Click image to enlarge

 

Technical Specifications

  • 3 laser, separate interrogation of sample for each laser
  • 16 color detection + 2 scatter parameters (from 488nm)
  • Hydrodynamically focused sample interrogated within a quartz cuvette flow cell
  • Sample aspiration rates at 12ul, 35ul, 60ul per minute
  • 262,144 channel (18 bit) dynamic range on all parameters
  • PC Windows 7, Diva version 8.0.1
  • (HTS ready, has internal components allowing a HTS to be connected, HTS software option enabled)

Excitation Sources

  • 488nm 50mw, Coherent Solid State (Sapphire)
  • 640nm 40mw, Coherent "Cube" Laser system
  • 405nm 50mw, Coherent "Cube" Laser system

Emission Detected

Click image to enlarge

 

Technical Specifications

  • 4 laser, separate interrogation of sample for each laser
  • 18 color detection + 2 scatter parameters (from 488nm)
  • Hydrodynamically focused sample interrogated within a quartz cuvette flow cell
  • Sample aspiration rates at 12ul, 35ul, 60ul per minute
  • 262,144 channel (18 bit) dynamic range on all parameters
  • PC Windows 7, Diva version 8.0.1

Excitation Sources

  • 488nm 50mW, Coherent Solid State (Sapphire)
  • 640nm 40mW, Coherent "Cube" Laser system
  • 405nm 50mW, Coherent "Cube" Laser system
  • 355nm 60mW, Coherent “Genesis” laser system

Emission Detected

Click image to enlarge

 

Technical Specifications

  • 4 laser, separate interrogation of sample for each laser
  • 18 color detection + 2 scatter parameters (from 488nm)
  • Hydrodynamically focused sample interrogated within a quartz cuvette flow cell
  • Sample aspiration rates at 12ul, 35ul, 60ul per minute
  • 262,144 channel (18 bit) dynamic range on all parameters
  • PC Windows 7, Diva version 8.0.1

Excitation Sources

  • 488nm 50mW, Coherent Solid State (Sapphire)
  • 640nm 40mW, Coherent "Obis" Laser system
  • 405nm 50mW, Coherent "Obis" Laser system
  • 355nm 60mW, Coherent “Genesis” laser system

Emission Detected

Click image to enlarge

 

Technical Specifications

The Beckman Coulter GALLIOS is equipped with four excitation lasers (405, 488, 561 and 635 nm). This is a 10-color, 12-parameter instrument with automatic sample loading for up to 32 tubes. The instrument operates on Kaluza for Gallios software (version 1.0).

Emission Detected

488nm Blue Laser
  • FSC
  • SSC
  • FL1: 525nm, FITC/Alexa 488/GFP
  • FL2: 575nm, PE
  • FL3: 620/30nm, PE-TxRd
  • FL4: 695/30nm, PE-Cy5, PerCP-Cy5.5
  • Alternative FL4: 675nm, PerCP (filter change)
  • FL5: 755nmLP, PE-Cy7
561nm Yellow-green Laser/ coaxial with 488nm:
  • FL2: 575nm, dsRed
  • FL3: 620/30nm, mCherry, RFP
635nm Red Laser:
  • FL6: 660nm, APC
  • FL7: 725/20nm, Alexa-700
  • FL8: 755nmLP, APC-Cy7
405nm Violet Laser:
  • FL9: 450/50nm, Pac Blue, BV421
  • FL10: 550/40, AmCyan, BV510

Alternative Filters

FLUOROCHROME Filter Available
BUV395 379/28
BUV737 740/35
BV605 610/20
BV650 660/20
BV711 710/50
Calcium Flux 405/20
Side Population 645LP
YFP 546/20
PerCP 670/LP

A Becton Dickinson LSR flow cytometer was modified to include a small 785-nm IR diode laser with 44-mW output power in addition to a 405-nm solid-state laser, a 488-nm air-cooled argon laser, and a 658-nm solid-state laser. 

 

The ZetaView small particle analyzer employs Nanoparticle Tracking Analysis (NTA) which relates the rate of Brownian motion of the particles in liquid suspension to particle size. The rate of movement is related to the viscosity and temperature of the liquid; it is not influenced by particle density or refractive index. NTA allows the determination of a size distribution profile of small particles with a diameter of approximately 10-1000 nanometers (nm) in liquid suspension. Additionally, the ZetaView is also capable of measuring the so-called zeta-potential of the particles which is a measure of the electric charge on the nanoparticle surface.

Our facility has two ZetaView analyzers which share a single flow cell module which is interchangeable between the two analyzers. One analyzer is equipped with a 488nm laser while the other is equipped with 405nm laser. For particle size, concentration and zeta potential measurements, both analyzers can be used interchangeably. When detection of fluorescent particles is desired, the analyzer of choice needs to match the excitation spectrum of the intended fluorochrome to be detected.

The primary application of the NTA analysis at Roswell Park is in the field of extracellular vesicles (EV) research where NTA is the accepted method for determining size distribution and concentration of EVs. Exosomes are cell-derived vesicles with a diameter between 30 and 100 nm. Evidence is accumulating that exosomes have specialized functions and play a key role in intercellular communication and can potentially be used for prognosis, for therapy, and as biomarkers for health and disease. Amongst the many detailed studies of exosomes that can include studying their phenotype and cargo, measurement of their size and concentration are additional key parameters in the context of their biological function.

The ZetaView instruments are QC’d prior to reservations and are available 24/7 to trained users by reserving time on the FOM calendar. 

 

FLUOROCHROME Filter Available
BUV395 379/28
BUV737 740/35
BV605 610/20
BV650 660/20
BV711 710/50
Calcium Flux 405/20
Side Population 645LP
YFP 546/20
PerCP 670/LP