Flow and Image Cytometry Shared Resource (FICSR)

Cell sorting

The Flow and Image Cytometery Shared Resource (FICSR) houses three cell sorter instruments in a BSL2+ facility. Two of these are available for operator-assisted sorting, each with a slightly different configuration, but otherwise similar capabilities.

The BD Aria I, upgraded in 2010, is equipped with 12 fluorescent PMTs and 405, 488, 561, and 640 nm lasers.

The Aria II is equipped with four lasers (355nm, 405nm, 488nm, and 633nm) and has 13 fluorescent detectors. Both instruments have the capacity to perform four-way sorts, as well as sorts into plates and onto slides. Both instruments are placed in a Bio-Bubble biocontainment system and have additional aerosol containment capabilities.

The Aria instruments are maintained and operated by dedicated facility staff members and are available during regular business hours by reservation. In a typical workflow, researchers will bring their stained single-cell suspensions to FICSR along with controls, buffers, and collection tubes. Then, together with the operator, the experiment is set up and sort logic is defined. After setup, researchers will not need to be present for the remainder of the sort duration. There are training options for individuals who would like to run these sorters unassisted.

A third, more user-friendly flow cytometry sorter, the SONY MA900, is available for unassisted use 24/7 following training by our staff. This instrument is equipped with 12 fluorescent PMTs and 405, 488, 561, and 638 nm lasers, and is placed in a biosafety cabinet. The instrument has the capacity to do four-way tube sorts as well as sorts into plates.

The sorter schedules can be found on our Facility Online Manager (FOM) calendar, and an active FOM account is required for access to the sorters. Reservations for the Aria sorters can only be scheduled by the FICSR sorter operators.

For sorts that have not been performed previously, please submit the online Sort Information form to request time. The operators will contact you to confirm your appointment and to discuss further specifics. Investigators new to sorting or embarking on a new project are encouraged to discuss their goals with FICSR staff prior to scheduling time.

If you are requesting a repeat of a sort that has been done before in our facility, a short Sort Appointment Request form can be submitted. The FICSR staff will contact you to finalize the appointment details. Appointments are not guaranteed until confirmed and entered in FOM.

Reservations for the SONY sorter can be placed on FOM by approved users after submission of the Sort Information Form to relay safety concerns.

Detailed information pertaining to our operation routine, reservations, cancellation policies, sample requirements, and collection options can be found in our document “Guidelines to Sorting”.

In addition to flow sorting capabilities, our facility also hosts a Miltenyi Classic AutoMACS for automated magnetic cell sorting. Reservations for this instrument are available to all trained researchers through FOM.

Contact Information: 

Orla Maguire: 716-845-5890
Xiaojun Liu: 716-845-1745  
Roswell Park Campus Location: Cancer Cell Center, Room C315

Cell sorting instrumentation

Our BSL2+ room in the Cancer Cell Center, Room C315, houses the two BD Aria cell sorter instruments and the SONY MA900.

The BD Aria I was upgraded in 2010 and is equipped with 12 fluorescent PMTs over four lasers (405 nm violet, 488 nm blue, 561 nm yellow-green, and 640 nm red). This configuration is similar to the LSRII B analyzer, but with less options for the violet laser. The instrument operates with a 100-micron nozzle/20 PSI or 70 micron nozzle/35 PSI configuration. Detailed specifications can be found below. The Aria I is placed in a Bio-Bubble negative-pressure biosafety cabinet that provides the BSL2+ environment to protect the operator.

The BD Aria II is equipped with four lasers (355 nm UV, 405 nm violet, 488 nm blue, and 633 nm red) and has 13 fluorescent detectors. This configuration is similar to the Fortessa B analyzer, but with less options for the violet laser. Current options for configuration on the Aria II are 100/20; 85/45; 85/30; 70/35, and 130/10 (nozzle size/PSI). Detailed specifications can be found below. The Aria II is placed in a Bio-Bubble negative-pressure biosafety cabinet that provides the BSL2+ environment to protect the operator.

The SONY MA900 was installed in 2019 and is equipped with 2 coaxial laser combinations, 488/561 nm and 405/638 nm, for detection of up to 12 fluorescent colors. The instrument operates with sorting chips that come in 70, 100 and 130-micron nozzle sizes, and setup is automated so that the user requires minimal training. Further detailed specifications can be found below.

All instruments have the capacity to do one- to four-way sorts into a variety of tube styles, such as 12x75 mm tubes of any material, Eppendorfs, PCR tubes and 15-ml conical tubes (two-way only). They can also sort into plates (6, 12, 24, 48, 96, and 384 wells).

Our cell sorters undergo a stringent routine maintenance and cleaning protocol to warrant aseptic sorting conditions at all times. We use sterile-filtered HEPES-buffered saline with EDTA as sheath fluid.

For magnetic cell sorting, our facility houses a Miltenyi Classic autoMACS. This instrument is located in a biosafety cabinet in the Cancer Cell Center, Room 322, and does not require operator assistance. Columns, clean bottles, and cleaning solution are provided. The user is responsible for running buffer and beads. Daily usage charges apply.

Cell imaging services

Confocal microscopy

Our Leica TCS SP8 Laser Scanning Spectral Confocal Microscope was put in operation in early 2018. The confocal has four laser lines (405nm, 488nm, 552nm, and 638nm) and is equipped with 10X dry, 20X dry, and 63X oil immersion objectives. Further specifications are detailed below.

First-time users of the Leica TCS SP8 confocal microscope must contact the principal operator Meghana Bapardekar at 716-845-3470 to schedule a new user training session (~45 minutes) before working independently on the microscope. All users should reserve time on the microscope using the FOM calendar system. Operator assistance is available by scheduling an appointment during regular business hours.

Live cell imager

The Leica AF6000LX live cell imaging system enables time-lapse videography of cell cultures in a temperature- and CO2-controlled environment. The microscope stage is adaptable to receiving multi-well plate formats as well as slide-based formats (including multi-well chamber slides). The software enables multiple user-defined imaging positions, as well as user-defined imaging time intervals.

In addition to polarized bright field imaging, the instrument has B/G/R fluorescent imaging capabilities using a mercury short-arc lamp excitation source. For multiple position imaging, the microscope is equipped with long-working distance 10x, 20x and 40x objectives.

A 63x objective is also available but due to the requirement of immersion-oil, this magnification is not suitable for multiple positions if the positions require relatively large stage movement or for long time periods of imaging. See details below.

The Live Cell Imager is available by appointment only. To make an appointment please contact Hans Minderman at 716-845-1162.

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