Instrumentation

Two fully-configured BD SORP FACSAria cell sorters, “Jupiter” and “Neptune”

FACSAria “Jupiter” and FACSAria “Neptune” are 20-parameter, 18-color cell sorters equipped with 5 high-powered, spatially-separated laser lines (355, 405, 488, 561, and 640

Please see the CSCI FACSAria Filters and Fluorophore Guide for more details about Jupiter and Neptune’s configurations.

FACSAria Laser Lines (adjustable power):

  • 355 nm – 60 mW
  • 405 nm – 100 mW
  • 488 nm – 100 mW
  • 561 nm – 100 mW
  • 640 nm – 140 mW

Note that the configurations of both “Jupiter” and “Neptune” are identical, facilitating portability of experiments between the two instruments.

FACSAria features include:

  • Highly stable fixed-alignment optics that greatly simplify setup and troubleshooting
  • Next-generation square cuvette which offers superior fluorescence sensitivity compared to sense-in-air sorters and superb recovery performance for neurons and other large cells
  • Four nozzle sizes - 70 μm, 85 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
  • Sorting onto a variety of plate types, including 96-well, 384-well, and Terasaki plates
  • Cooling 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 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.”

Bio-Rad S3e cell sorter: “Juno”

Bio-Rad S3e "Juno" is a two-laser, four-color benchtop fully-automated, walk-away cell sorter that boasts a variety of features that enable unprecedented ease of use. New users can be trained to operate the instrument in far less time than would be required for other cell sorters.

"Juno" is equipped with two colinear lasers (adjustable power):

  • 488 nm (blue), 100 mW
  • 561 nm (yellow-green), 100 mW

This instrument's optical detectors are configured as described below:

  • FL1 - FITC, GFP (488 nm excitation only)
  • FL2 - PE, dsRed (both 488 nm and 561 excitation)
  • FL3 - mCherry, PE-Texas Red (both 488 nm and 561 excitation)
  • FL4 - PE-Cy5, PE-Cy7, PerCP-Cy5.5, 7-AAD, APC, OR APC-Cy7 (both 488 nm and 561 excitation)

"Juno" is extremely well-suited to simple sorts of fluorescent proteins or cell-surface markers of up to 4 colors.

Key Features:

  • Sense-in-air cell sorting technology
  • Fully-automated, one-button startup
  • Fully-automated, one-button quality control that includes auto-alignment of lasers via picomotor control
  • Automatic, motorized alignment
  • Fully-automated, one-button shutdown
  • Sort volume monitoring
  • Stream monitoring for a fully "walk-away" experience
  • Sample line bubble detector - entire volume of sample can be sorted
  • Sample input and output cooling
  • Sorting into 5 or 1.5 mL tubes

Important: “Juno” is housed in a Class I biosafety cabinet, so human tissue can be sorted on this system.

Bio-Rad ZE5 cell analyzer, “Apollo”

The Bio-Rad ZE5 “Apollo” is 30-parameter fully-automated flow cytometer cell analyzer equipped with five lasers and can measure 27 fluorescence parameters simultaneously, 488 nm forward scatter, 405 nm forward scatter (optimized for smaller cells and particles), and side scatter (488 nm). The ZE5’s unique optical design offers superior fluorescence measurement sensitivity for better resolution of populations.

Please see the CSCI ZE5 “YETI” Analyzer Configuration for details of specific filter and laser combinations.

Please see the CSCI ZE5 Fluorophore Guide for more information for panel design on the ZE5.

The ZE5 was designed with maximum flexibility in mind. Samples can be acquired from virtually any type of tube or plate type, and new plates can be quickly calibrated through Everest software. Acquisition is fully-automated, so users can set up the experiment and walk-away while it is running. This instrument implements sophisticated monitoring tools, including a bubble detector, so the instrument will stop acquisition automatically if a problem is encountered.

Additionally, the ZE5 is capable of acquisition speeds that far surpass those of other analyzers. The state-of-the-art electronics can process up to 100,000 events per second with no electronic aborts, and the sensitivity of the system permits sheath pressure of 10 PSI, approximately double that of comparable analyzers, which facilitates higher sample throughput. When using High-Throughput Mode, a 96-well plate can be acquired in less than 10 minutes at the fastest settings.

Main features include:

  • Simultaneous measurement of up to 27 colors
  • Two forward scatter detectors, including one measured off of the 405 nm laser and optimized for small particle detection
  • Sampling for any type of tube. Up to 40 5mL tubes can be loaded onto the instrument at once for walk-away analysis
  • Fully-automated startup and shutdown processes
  • Fully-automated sample line cleaning and unclogging
  • On-board room temperature-stable quality control beads and an automated one-button QC feature
  • Intuitive wizard-based experiment setup tool with integrated spectra viewer
  • Sample temperature control
  • Sample tube agitation
  • Sample line clog monitoring
  • Virtually zero dead volume
  • State of the art reduced-pulse peristaltic pump that facilitates fine control of sample rate, volumetric sampling and return of unused portion of sample to tube or well
  • Probe crash sensing mechanism to prevent bending of sample probe if path is obstructed
  • High-throughput mode for extremely rapid acquisition
  • Automated software-based plate calibration tool

BD FACSCelesta analyzer, “Pluto”

The BD FACSCelesta "Pluto" is 14-parameter, 12-color analyzer equipped with three lasers.

The FACSCelesta offers high-sensitivity for up to 12-color panels and is controlled operated with FACSDiva software, facilitating an easy transition for users from cell analysis to cell sorting on the FACSAria instruments.

Please see the CSCI FACSCelesta Configuration Guide for further details of the configurations.

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 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 a 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

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 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, 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.

Luminex-200

The Luminex-200 is a magnetic microsphere-based multiplexed assay system. This instrument is flow cytometry-based and can detect up to 80 targets in a single tube. The Luminex can detect both protein and nucleic acid targets and can read a 96-well plate in approximately 45 minutes.

Fluidigm BioMark HD microfluidic multiplexed genomic analyzer

The Fluidigm BioMark HD is a microfluidics-based multiplexed genomic analyzer that can accommodate a wide variety of sample types and chemistry. This instrument is capable of sensitivity of 15 orders of magnitude to enable measurement of gene expression profiles of single cells.

Applications include:

  • Gene expression
  • Genotyping
  • Protein expression
  • Digital PCR
  • Sample identification

The CSCI Flow Cytometry BioMark is capable of running both the 48.48 and 96.96 chips.

The 48.48 chip contains 48 assay inlets and 48 sample inlets for a total of 2,304 reaction chambers of 10.1 nL. The 96.96 chip contains 96 assay inlets and 96 sample inlets for a total of 9,216 reaction chambers of 6.7 nL.

Applied Biosystems QuantStudio 7 Flex real-time qPCR system

The QuantStudio 7 Flex real-time PCR System delivers reliability, sensitivity, and accuracy with the versatility of four interchangeable blocks and is optimized to enable the broadest range of qPCR applications.

Available blocks are:

  • 96-well
  • 96-well fast
  • 384-well
  • TaqMan® Array card (384-well micro fuidic card)

Note: CSCI Flow Cytometry currently has only the 96-well and 384-well blocks.

The QuantStudio Flex 7 can measure up to 6 colors with 21 filter combinations.

Kep applications include:

  • Gene expression
  • miRNA profiling
  • SNP genotyping
  • Copy number variation
  • Protein thermal shift
  • High resolution melt
  • Pathogen detection
  • Pharmacogenomics