The Flow Cytometry Shared Resource is located the Rosenstiel Medical Science Building, room 7147 (RMSB 7147). The shared resource provides the following instrumentation:

• BD FACS Canto-II: cell analyzer with 8 fluorescent parameters and 2 light-scatter parameters.
• BD LSR-Fortessa-HTS: cell analyzer with 18 fluorescent parameters and 2 light-scatter parameters.
• BD FACSymphony A5 SE: cell analyzer with 30+ fluorescent parameters and 2 light-scatter parameters using both conventional compensation and spectral unmixing platforms.
• Cytek Aurora: cell analyzer with 40+ fluorescent parameters and 3 light-scatter parameters using the spectral unmixing platform.
• BD FACS AriaII: BSL-1 cell sorter with 13 fluorescent parameters and 2 light-scatter parameters.
• BD FACS Aria Fusion: BSL-2 cell sorter with 15 fluorescent parameters and 2 light-scatter parameters.
• BD FACS AriaIIu: BSL-2 cell sorter with 17 fluorescent parameters and 2 light-scatter parameters.
• Beckman Coulter CytoFLEX SRT: BSL-2 cell sorter with 15 fluorescent parameters and 2 light-scatter parameters available for self-operated cell sorting 24/7/365.
• Standard BioTools Inc. (formerly Fluidigm) Helios CyTOF Mass Cytometer.
• Standard BioTools Inc. (formerly Fluidigm) Hyperion Imaging Mass Cytometer.
• Zeiss fluorescence microscope
• Invitrogen Countess cell counter

Use of the FCSR’s instruments

The facility offers virtual and hands-on training sessions to individual users for self-operated use on all the cell analyzers (FACS Canto-II, LSR-Fortessa-HTS, FACSymphony A5 SE and Cytek Aurora), with operator assistance available upon request for all users (trained and untrained). All three Becton Dickinson cell sorters (FACS AriaII, FACS AriaIIu, FACS Aria Fusion) are operator-assisted, with the CytoFLEX SRT cell sorter available as an operator-assisted or self-operated instrument. Self-operated cell sorting on the CytoFLEX SRT requires comprehensive training offered by the FCSR. The CyTOF Helios Mass Cytometer and Hyperion Imaging Mass Cytometer are operator-assisted. Experience and trained internal users can request 24/7/365 access to all self-operated instruments, which will be granted after demonstrating proficiency in proper instrument operation and shutdown.

Instruments

Analysis

• BD FACS Canto-II Analyzer – The FACS Canto-II is a 3-laser flow cytometric cell analyzer with 8 fluorescent parameters and 2 light-scatter parameters.Using FACSDiva 9 acquisition software on a PC workstation.
• BD LSR-Fortessa-HTS Analyzer – The LSR-Fortessa-HTS is a 5-laser flow cytometric cell analyzer with 18 fluorescent parameters and 2 light-scatter parameters.Equipped with a high-throughput automated sample loader for rapid analysis of stained cells directly from a 96-well plate.Using FACSDiva 9 acquisition software on a PC workstation.
• Cytek Aurora, Spectral Analyzer – The Aurora is a 5-laser flow cytometric cell analyzer with 64 fluorescent detectors and 3 light-scatter parameters capable of analyzing up to 40+ fluorescent parameters using the spectral unmixing platform.Equipped with an Automatic Micro-Sampling System for rapid analysis of samples directly from a 96-well plate.Each fluorochrome has a unique spectral signature across the full fluorescence emission spectrum, as a spectral analyzer, the Aurora can separate reagent combinations that were previously unresolvable due to spectral interactions. Using SpectroFlo acquisition software on a PC workstation.
• BD FACSymphony A5 SE Analyzer – The FACSymphony A5 SE is a 5-laser flow cytometry cell analyzer with 48 fluorescent detectors and 2 light-scatter parameters capable of analyzing 30+ fluorescent parameters using both conventional compensation and spectral unmixing platforms.Instrument features an optimized filter set to collect the full spectrum of emitted light allowing for simultaneous analysis of fluorochromes with similar spectral signatures.Using FACSDiva 9 acquisition software on a PC workstation.

Sorting

Becton Dickinson Cell Sorters – The three FACS Aria cell sorters are used for complex analysis and sorting of cell samples. They incorporate a fixed-alignment flow cell for enhanced sensitivity of fluorescent detection and ease of optical alignment. All have aerosol containment systems and use FACSDiva 8 acquisition and sorting software on a PC workstation.

• BD FACS Aria Fusion – The FACS Aria Fusion is a 4-laser high-speed cell sorter with 15 fluorescent parameters and 2 scatter parameters.Equipped with sample temperature control and an Automatic Cell Deposition Unit (ACDU), for sorting directly into 96-well plates for tissue culture and single cell cloning.Instrument includes an integrated bio-safety cabinet for processing on hazardous samples up to BSL-2+ classification.
• BD FACS Aria-IIu – The FACS AriaIIu is a 5-laser high-speed cell sorter with 17 fluorescent parameters and 2 scatter parameters.Equipped with sample temperature control and an Automatic Cell Deposition Unit (ACDU), for sorting directly into 96-well plates for tissue culture and single cell cloning.Housed in a Baker BioProtect-II bio-safety cabinet for processing of hazardous samples up to BSL-2+ classification.
• BD FACS Aria-II – The FACS AriaII is a 4-laser high-speed cell sorter with 13 fluorescent parameters and 2 scatter parameters.Equipped with sample temperature control and an Automatic Cell Deposition Unit (ACDU), for sorting directly into 96-well plates for tissue culture and single cell cloning.This instrument is strictly used for non-hazardous samples classified as BSL-1.

Beckman Coulter Cell Sorter

• CytoFLEX SRT – The CytoFLEX SRT is a 4-laser cell sorter with 15 fluorescent parameters and 2 scatter parameters.Equipped with sample temperature control and a CyClone movement system, for sorting into tubes, slides or multiwell plates.Housed in a bio-safety cabinet for processing of hazardous samples up to BSL-2+ classification. Using CytExpert SRT acquisition software on a PC workstation, the CytoFLEX is designed for ease-of-operation.

Mass Cytometry

Standard Biotools (Formerly Fluidigm) Mass Cytometry

• CyTOF Helios (Suspension Mass Cytometry) – The CyTOF Helios Mass Cytometer analyzes single cell suspensions labeled with antibodies conjugated to heavy metal isotopes (50+ distinct metal isotopes).The cells are nebulized and sent through argon plasma, which results in an ion cloud.This ion cloud is processed, filtered and then detected in a time-of-flight mass spectrometer.Using CyTOF Software 7.0 on a PC workstation.
• CyTOF Hyperion (Imaging Mass Cytometry) – The Hyperion Imaging Mass Cytometer analyzes FFPE or frozen solid tissue sections or fixed cells that are adhered to a slide and labeled with up to 50+ antibodies conjugated to heavy metal isotopes.A UV laser ablates the tissue of interest in 1 micron square increments (“pixels”) and ionized and detected in a time-of-flight mass spectrometer.Multiplexed images are generated at subcellular resolution.Using CyTOF Software 7.0 on a PC workstation.

Other

• Zeiss fluorescence microscope – Used for the examination of fluorescent cells, before and/or after cell sorting.
• Invitrogen Countess cell counter – Used for cell counting with trypan blue dye.Only 10ul of sample is needed to determine total cell count and cell viability.
• Data analysis software – Site licenses for FlowJo, FCS Express and TerraFlow Visiopharm available with annual fees per license or per use.Visiopharm AI-assisted image analysis for Hyperion imaging mass cytometry data, on a per image basis. Data analysis is available for certain applications on a fee-for-service basis.