Instrumentation

CSCI Flow Cytometry maintains two 20-parameter, 18-color cell FACSAria instruments, “Jupiter” and “Neptune”,  equipped with 5 high-powered, spatially-separated laser lines. The CSCI Flow Cytometry FACSAria cell sorters are reliable workhorse machines that consistently isolate cells of high purity, recovery and viability.

FACSAria features include:

• Highly stable fixed-alignment optics which greatly simplify setup and troubleshooting
• Next-generation square cuvette which offers superior fluorescence sensitivity and superb recovery performance for cells of all sizes, including large and fragile cells
• Three nozzle sizes - 70 μm, 100 μm, and 130 μm to accommodate cell types of various sizes and robustness
• 4-way sorting into a variety of tube types, including 1.5 mL, 5 mL, and 15 mL tubes (2-way sorting only into 15 mL tubes)
• Direct deposition into a variety of plate types, including 96-well, 384-well, and Terasaki plates
• Temperature control of both sample input and sorted fraction
• Integrated bubble detector on sample path, allowing sorting of entire volume sample without the worry of introduction of air bubbles into the sample fluidics
• Aerosol Management Systems (AMS) for biosafety concerns

Important: “Neptune” is equipped with a “BioBubble” Class I biosafety cabinet, while Jupiter” is not. All sorting of primary human tissue must be performed using “Neptune.”

CSCI Flow Cytometry maintains two Sony MA900 cell sorters: "Mars" and "Venus". The Sony MA900 is a benchtop cell sorter equipped with 4 partially spatially-separated lasers (405 nm, 488nm, 561 nm, 638 nm) and is capable of measuring 14 optical parameters (12 fluorescence channels and 2 light scatter channels). The Sony is built around its highly innovative and proprietary sorting chip, which combines microfluidics with traditional electrostatic droplet sorting. This technology facilitates a degree of automation and ease-of-use not offered by any other cell sorter. Sorting chips are available in 100, 130 and 70 um sizes with associated pressures to accommodate a wide variety of cell types. The MA900 can simultaneously collect up to 4 unique cell populations into tubes (1.5, 5, and 15 mL) and is capable of direct deposition down to the single-cell level into up to 384-well plates.

Fully automated startup and maintenance features and intuitive software facilitate and extremely high level of usability for independent operation

• Three nozzle sizes: 100, 130 and 70 μm
• Proprietary sorting chip for ease of use and stability
• 4-way sorting into 1.5 mL, 5 mL, and 15 mL tubes
• Direct plate deposition down to single cells into any plate type up to 384-wells
• Temperature control of both sample input and output
• Demonstrated high performance in terms of purity, recovery and viability
• Housed in a Class II biosafety cabinet for sorting of primary human tissue

The BD FACSDiscover S8 Image-Enabled Spectral Cell Sorter is the first instrument that is capable of cell sorting based on imaging data. Patented CellView™️ high-speed imaging technology facilitates spatially resolved signal measurements without a camera by using the optical and electronic components available on flow cytometers.

In addition to image-enabled sorting, the FACSDiscover S8 incorporates a spectral detection system based on avalanche photodiodes (ADPs) measuring fluorescence from 5 laser lines for true high parameter sorting.

Here are some of the unique applications made possible by the FACSDiscover S8.

1. Image-Enabled Sorting: Utilize quantified imaging data (“features”) in your sort logic. Imaging features can be plotted and gated like traditional flow cytometry data and used for sorting
   • Differentiation of punctate from diffuse fluorescence signal
   • Detection of the degree of colocalization of two fluorescence signals
   • Measurement of the distance between two fluorescence signals
   • Detection of cell-cell interactions
   • Enhanced doublet exclusion
   • Sorting cells based on cell size
2. High parameter spectral cell sorting of panels of at least 50 colors*.
3. Six-way sorting to isolate up to six populations simultaneously from a sample.
4. Single cell deposition directly onto plates, including 384-well plates.
5. Index sorting of both imaging and flow cytometry data when sorting into plates.
6. 85, 100, and 130 μm nozzles to accommodate a variety of sample types.
7. Biosafety cabinet for sorting of primary human tissue.

* Konecny AJ, Mage PL, Tyznik AJ, Prlic M, Mair F. OMIP-102: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells. Cytometry A. 2024 Jun;105(6):430-436. doi: 10.1002/cyto.a.24841. Epub 2024 Apr 18. PMID: 38634730; PMCID: PMC11178442.

You can find a breakdown of the FACSDiscover's features below:

Image-Enabled Sorting

Imaging Specifications:

1. Six Imaging Channels:

   • 3 morphological/non-fluorescence images (forward scatter, side scatter, and light loss​

   • 3 fluorescence images

     • FL1: LP505, 534/46 (FITC, AF488, GFP, YFP, etc)

     • FL2: LP570, 600/60 (PE, tdTomato, RFP, mScarlet, RY586, etc.)

     • FL3: LP675, 788/225 (DRAQ5, PE-Cy7, RB705, RB780)

2. Magnification: ~15x
3. Resolution: 1.5 μm^2/pixel
4. Field of View: 60 μm

Imaging Features

Imaging features permit quantification of imaging measurements for biological measurements. These quantified features can be plotted like flow cytometry data and used for sorting. The following features are currently available on the FACSDiscover S8:

• Light Loss
• Forward Scatter
• Side Scatter
• Correlation
• Delta Center of Mass
• Diffusivity
• Eccentricity
• Max Intensity
• Radial Moment
• Center of Mass (X)
• Center of Mass (Y)
• Long Moment
• Short Moment
• Size
• Total Intensity

Extensive information details are available about each of these features. Please contact the core facility if you are interested in learning more.

Spectral Cell Sorting

The FACSDiscover S8 is equipped with a state-of-the-art spectral detection system coupled with BD patented SpectralFX™️ electronics and algorithms.

Highlights of SpectralFX™ include:

1. Full spectrum optics and optimized hardware design with 78 solid state detectors and 5 lasers for all the classical benefits of spectral flow cytometry.
2. System aware unmixing algorithm adapts to your sample and instrument in real time to manage spread.
3. Next generation QC system uses LEDs and beads to measure noise, perform gain calibration and provide real time noise/signal hardware information.
4. Guided workflow allows users to learn quickly and use best practices in experimental setup.

Spectral Configuration

The FACSDiscover S8’s SpectralFX™ technology is equipped with the following hardware components for spectral detection.

1. Five spatially separated excitation lasers
   • 349 nm (UV), 30 mW
   • 405 nm (violet), 50 mW
   • 488 nm (blue), 100 mW (488 nm laser is split between the SpectralFX and CellView systems)
   • 561 nm (yellow green), 50 mW
   • 637 nm (red), 100 mW
2. 78 fluorescence avalanche photodiode (ADP) photodetectors
   • 349 nm: 22 detectors, 365 - 860 nm
   • 405 nm: 20 detectors, 410 - 860 nm
   • 488 nm: 16 detectors, 495 - 860 nm
   • 561 nm: 12 detectors, 570 - 860 nm
   • 637 nm: 8 detectors, 645 - 860 nm

The Sony ID7000 "Sirius" Spectral Flow Cytometer is a cutting-edge spectral flow cytometer analyzer equipped with 5 lasers and 147 fluorescence photodetectors, giving the instrument the ability to detect more than 40 fluorochromes in a single sample. The full spectrum detection capabilities of the instrument permit measurements of virtually any excitable fluorochrome without dedicated filter sets. In addition, by measuring spectral signatures of each fluourochrome, the ID7000 can detect the presence of nearly overlapping dyes. Furthermore, autofluorescence can be treated as a unique cellular parameter and subtracted from total fluorescence, reducing background and increasing resolution.

At the heart of the ID7000 is the best-in-class auto-loader capable of true hands-free sample

acquisition from a variety of plate types, including both 96 and 384-well formats, and a multi-tube rack. Temperature control and sample mixing are integrated into the auto-loader and can be programmed and tailored uniquely to each experiment. Intuitive ID7000 Software simplifies the workflow for setting up a high parameter experiment, collecting data, and

accurately performing spectral unmixing.

Excitation: (all lasers are spatially separated)

• 355 nm
• 405 nm
• 488 nm
• 561 nm
• 637 nm

Detection:

• 147 fluorescence detectors (multichannel and single channel PMTs) spanning the visible spectrum
• Light is partitioned by wavelength for spectral detection using custom diffraction grating technology
• Detection is apportioned per laser as described below:
  • 355 nm laser: 35 independent detectors (three single-channel PMTs + one 32 channel PMT)
  • 405 nm laser: 35 independent detectors (three single-channel PMTs + one 32 channel PMT)
  • 488 nm laser: 32 independent detectors (32-channel PMT)
  • 561 nm laser: 26 independent detectors (multichannel PMT)
  • 637 nm laser 19 independent detectors (multichannel PMT)
• Forward and side scatter (488 nm)

The NovoCyte cell analyzer platform is a flexible reliable conventional flow cytometer workhorse analyzer suitable for a variety of applications. Controlled by extremely well-designed and powerful NovoExpress software, the NovoCyte is a robust, easy-to-use workhorse analyzer platform capable of handling a range of experiments, including those requiring measurement of at least 20 colors. Equipped with high-powered lasers and unique silicon photomultiplier (SiPM) detectors that require only minimal gain adjustments and facilitate a 7.2-decade log scale, the NovoCyte is capable of sensitive and consistent measurements.

Key features include:

• Up to 5 high-power Coherent lasers (349 nm (Penteon only) 405 nm, 488 nm, 561 nm, and 640 nm)
• Up t0 32 low-noise, highly efficient Hamamatsu silicon photomultiplier (SiPM) SiPM detectors
• 7.2-log decade scale precluding the need for extensive detector gain adjustment
• Flexible universal sample loader (NovoSampler Q) for tubes and plates, including 40-tube rack for 5 mL and 1.5 mL tubes
• Stable syringe pump-driven sampling system that is capable accurate absolute counting (particles/uL) and return of unused sample
• Full automation of routine tasks - startup, QC, cleaning, and shutdown for walk-away and worry-free operation
• Automatic unclogging
• Extremely user-friendly and richly featured software that has full analysis capability as well as built-in modules for specific applications, including cell cycle and proliferation.

You can find the NovoCyte optical configuration here.

AmnisⓇ ImageStreamX Mk II Imaging Cytometer

The ImageStream®X Mk II Imaging Flow Cytometer combines the speed, sensitivity, and phenotyping abilities of flow cytometry with the detailed imagery and functional insights of microscopy. In addition to the fluorescence intensity values that are typically provided by flow cytometric measurements, the ImageStream also captures a 12-channel image of each cell, including brightfield, side scatter, and fluorescence, through camera-based detection of a single-cell suspension. Unique time-delay integration of signal attributes signals measured on a pixel-by-pixel basis among all 12 channels to each individual particle, facilitating extremely high-sensitivity measurements. The throughput afforded by the ImageStream’s flow cytometry-style measurement system provides statistical significance of large data sets and allows signal quantification and spatial information of rare populations. This unique combination enables a broad range of applications that would be impossible using either technique alone.

The CSCI Flow Cytometry ImageStreamX Mk II is fully-configured with the following features:

Four excitation lasers for fluorescence-based measurements

• 405 nm
• 488 nm
• 561 nm
• 642 nm

12 imaging channels across two proprietary CCD cameras

• Brightfield
• Side scatter
• 10 fluorescence channels

Three objective lenses:

• 60X
• 40X
• 20X

You can find the ImageStream optical configuration here.

AutoSampler for unattended 96-well plate acquisition. This sophisticated system provides the following benefits:

• In-line bubble detection for unattended acquisition
• Automatic process logging and error notification
• Automatic sample probe rinse between samples and <0.5% carryover
• Automatic sample resuspension via aspiration

The Extended Depth of Field (EDF) module keeps the depth of cells in focus without loss of sensitivity using Wavefront Coding™ technology from CDM Optics, which is a combination of specialized optics and unique image processing algorithms, to project more structures within the cell into one crisp plane of focus.

The ImageStreamX Mark II is engineered with a variety of sophisticated features that ensure robust performance and facilitate easy-of-use, allowing the machine to be operated by end users after basic training:

• Automated startup and shutdown procedures, including decontamination
• On-board fluidics and level monitoring
• Automated calibration and quality control which incorporates on-board SpeedBeadsⓇ and motorized optics to ensure consistent laser alignment. The automated calibration and QC perform a variety of sophisticated tests to ensure that the machine is operating at peak performance before sample acquisition.
• Automatic mixing of sample before acquisition
• Sample probe rinse between samples to prevent carryover (<0.5%)
• Sample return feature to facilitate virtually 100% sample utilization

Data analysis performed using IDEAS software, which is a powerful platform that incorporates both wizard-based and customized workflows to facilitate data analysis from any application.

Typical applications include but are not limited to the following:

• Cell Signaling (translocation)
• Internalization and phagocytosis
• Intracellular co-localization
• Shape change and chemotaxis
• Cell-cell interaction
• Cell death and autophagy
• Cell cycle and mitosis
• DNA damage
• Stem cell biology (differentiation)
• Microbiology
• Parasitology
• Exosome internalization
• Microparticle quantitation

IMPORTANT: Because the ImageStreamX MkII was purchased with funds awarded through the NIH S10 Shared Instrumentation Grant program (Award Number S10OD026845), publications utilizing data acquired with the ImageStream must acknowledge this grant. Please use the following language in publications in which you have used the ImageStream for your experiments:

Research reported in this publication using the ImageStreamX MkII imaging cytometer was performed in the Columbia University Stem Cell Initiative Flow Cytometry core facility at Columbia University Irving Medical Center and was supported by the Office Of The Director, National Institutes Of Health under Award Number S10OD026845. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

The Columbia Stem Cell Initiative Flow Cytometry core, in collaboration with the Columbia Genome Center Single Cell Analysis Core (SCC) is pleased to offer access to 10x Genomics single-cell technology.

After obtaining reagents from the Single Cell Analysis core, fully-trained users have independent access to the 10x Genomics Chromium-X controller, workspace, and associated equipment for the generation of barcoded cDNA from GEM-captured cells. Barcoded cDNA can then be taken to the SCC for library preparation or sequencing. Users also have the option to perform library preparation and arrange for sequencing independently.

Key Points:

• CSCI Flow Cytometry supports only the GEM-capture and cDNA barcoding steps. Library preparation and sequencing must be arranged with the SCC or independently by researchers.
• Users can obtain 10x GEM-X reagents from the SCC when using the core's library preparation and sequencing services.
• 10x Genomics access is offered on a self-use basis only. The service is recommended for researchers who require off-hours access to the Chromium Controller and plan to use the technology regularly. Researchers whose needs are infrequent are encouraged to utilize the SCC's full-service option.
• Users must complete comprehensive training with CSCI Flow Cytometry staff before being granted access to the Chromium Control and workspace.

Please see our CSCI Flow Cytometry 10x Genomics Service document for more details.

The BD Rhapsody™ HT Single-Cell Analysis System, coupled with the BD Rhapsody™ Scanner, allows flexible sample processing and cell capture from hundreds to hundreds of thousands of single-cells using a gentle and robust microwell-based cartridge technology and multitier barcoding system enabled by BD Rhapsody™ Enhanced Bead Technology. Multiple samples can be processed in a single run when utilizing BD multiplexing antibodies. The captured cellular information is utilized to generate various types of libraries for next-generation sequencing applications providing accelerated time to insight.

Highlights of the BD Rhapsody™ indclude:

Flexible cartridge design

• Up to 8 tests per cartridge
• Partial use of cartridge enables:
  • Running more or different types of experiments
  • Processing samples together or on different days

Low multiplet rate per lane

• 2.5% @ 10,000 cell load
• 5.2% @ 25,000 cell load
• 13.2% @ 55,000 cell load
• 20.9% @ 100,000 cell load

Minimal batch effects

• Consistent, reliable results with technical, biological, site-to-site and user-to-user replicates

Subsample beads

• Creates flexibility with experimental design
• Tool to measure sample quality
• Share beads across sites with collaborators

Archive beads

• Equivalent data obtained from fresh beads and stored beads (stable for up to 1 year at 4°C)
• Support flexible and collaborative workflow approach
• Backup for underperformed or failed library preps

CSCI Flow Cytometry maintains both 96-well and 384-well compatible CFX Opus Instruments

The CFX Opus is a robust and flexible real-time PCR platform with the following features:

• Optical measurements in up to 5 channels:
  • Channel 1: FAM (450-490 nm)
  • Channel 2: HEX (515-535 nm)
  • Channel 3: Texas Red (560-590 nm)
  • Channel 4: Cy5 (620-650 nm)
  • Channel 5: Quasar 705 (705-730 nm)
• Multiplexing for discrimination between up to five targets in a single reaction well
• Designated channel with an LED-filter photodiode combination for single-color FRET experiments (96-well unit only)
• LED shuttle optical system provides consistent optical measurements across your sample plate and precludes the need for calibration
• BR.io cloud platform for remote experiment setup, data retrieval and data analysis.
  • Please make sure to sign up for a BR.io account before utilizing the CFX.
• Free access to CFX Maestro feature-rich qPCR data analysis software for installation on users' personal computers.

Recommended Plate Types:

Bio-Rad recommends the following plate types for best results on the CFX platform:

For CFX Opus 384 systems:

Bio-Rad HSP3805 — Hard-Shell low-profile 384-well plates with clear shell and white wells

Bio-Rad HSP3865 — Hard-Shell low-profile 384-well plates with black shell and white wells

For CFX Opus 96 systems:

Bio-Rad HSP9655 — Hard-Shell low-profile 96-well skirted PCR plates with white shell and white wells 

The Cellometer Auto 2000 is a fluorescence-based cell and viability counter that uses bright field imaging and dual-fluorescence imaging via AO/PI (acridine orange/propidium iodide) method to quickly and accurately identify and count individual cells. Cell count, concentration, diameter, and % viability are automatically calculated and reported. The advantage of the AO/PI method is that it minimizes measurement interference by red blood cells, platelets or debris. Cell viability and count results can be obtained in 30 seconds.

The EVOS FLoid is an intuitive desktop 3-color fluorescent imaging system that was designed to the one of the simplest, fastest and easiest cell imaging systems.

Key Features:

• 3-color fluorescence and brightfield imaging with focus assist and adjustable focus, brightness, and contrast
• Speed: Images obtainable with a minute of startup - warmup, cool-down, or filter changes are not necessary
• Placement in a darkroom is not necessary due to the instrument's ability to block ambient light
• Long-life LED-based illumination
• Integrated reagent selection guide with protocols