Flow Cytometry Shared Resource (HICCC)

The Flow Cytometry Shared Resource (FCSR) is a joint operation between the Herbert Irving Comprehensive Cancer Center and the Columbia Center for Translational Immunology. The core director, Dr. Remi Creusot, oversees the daily operations of the FCSR and ensures that flow cytometry is an approachable research tool and within reach to all Columbia investigators. Dr. Creusot provides comprehensive and informative communication of the capabilities and applications of the Core; provides expert guidance, training, service and quality assurance through highly skilled and dedicated personnel; monitors user satisfaction; enables the design and performance of cost-effective flow cytometry experiments; and ensures that instruments and procedures are current and fulfill the needs of users in terms of capabilities and availability, and expands on new and innovative technologies as they become available. The FCSR provides: (1) multi-color panel flow data acquisition and analysis using flow cytometry analyzers, including a high-throughput system (HTS), (2) flow cytometry cell sorting (four-way or six-way or plate) using high speed cell sorters by dedicated operators of FCSR, including single cell index sorting, and (3) comprehensive consultation, panel design, hands-on training, troubleshooting, and data analysis services by PhD-level FCSR scientists. This broad and inclusive package of services enables investigators to analyze cell populations and their microenvironment. The FCSR has two locations: a site in the Irving Cancer Research Center, and an additional location in the Vagelos College of Physicians and Surgeons building with a collection of high-end flow analyzers and sorters.

Equipment

ICRC Site

  • BD LSR II; UV, violet, blue and red lasers; 20 detectors 
  • BD Fortessa; violet, blue, yellow and red lasers; 18 detectors 
  • BD Influx; violet, blue, yellow and red lasers; 17 detectors 
  • BD Influx; violet, blue, yellow and red lasers; 17 detectors
  • Leica DMI 6000B fluorescent microscope
     

Accessories

  • Analysis Workstation (ICRC 333)
     

VP&S Site

  • BD LSR II; UV, violet, blue, green, orange and red lasers; 20 detectors 
  • BD Fortessa; violet, blue and red lasers; 16 detectors 
  • BD Canto II; violet, blue and red lasers; 10 detectors 
  • BD Influx (BB/PS); violet, blue, yellow and red lasers; 17 detectors 
  • BD Influx (PS17-503); violet, blue, yellow and red lasers; 17 detectors 
  • Leica DMI 6000B fluorescent microscope
     

Accessories

  • BD High-Throughput System (connected to BD Fortessa)
  • BD High-Throughput System (available on demand) 
  • Analysis Workstation (PS17-503)
  • Analysis Workstation (BB1511)
     

The Flow Cytometry Shared Resource (FCSR)is located in two sites: the Vagelos College of Physicians and Surgeons Building (VP&S) and the Irving Cancer Research Center (ICRC).

Flow analyzers:  BD LSR II flow analyzers have the greatest capacity and allow detection of most commercially available fluorochromes, including the Brilliant series dyes (BUV, BV, BB), traditional organic dyes, and their tandems (FITC, PE, PerCP, APC, PE-TR, PE-Cy7, APC-Cy7), Alexa dyes (Alexa 488, Alexa 633, Alexa 700), and the inorganic QDot (Qdot 525, 605, 655, 705, 800). Both LSR IIs contain 18 photomultiplier tube (PMT) fluorescent detectors and 2 light scatter sensors and can analyze up to 20 parameters. The LSRII at the VP&S site has two additional lasers: 532nm (green) and 594nm (orange), which allow use of common microscopy dyes including Alexa532, 555, 594 (facilitating transfer of analysis from fluorescence microscopy to flow cytometry), as well as mCherry, and achieve better excitation of PE-containing dyes.

BD Fortessa flow analyzers are available in 3-laser or 4-laser configurations (405nm, 488nm, and 633nm, with or without 561nm). They are equipped with 14-16 fluorescent PMTs and two light scatter sensors for a total of 16-18 parameters, allowing for reasonably complicated studies that do not require UV dyes. One of them is connected to a High-Throughput System for automatic 96-well plate acquisition.

The BD Canto ll can measure up to eight fluorescent parameters and two light scatters.

The core also houses a Cytek Aurora spectral flow cytometer (UV, violet, blue, green and red lasers; 40+ parameters) owned by the Human Immune Monitoring Core. The FCSR provides maintenance and training for this instrument.

Flow cytometry sorters:  All FCSR cell sorters are contained in a certified biosafety hood in compliance with the NIH biosafety policy for sorting of unfixed human cells with aerosol containment in shared laboratory spaces. Similar to the LSR II and Fortessa analyzers, each sorter is configured with four lasers: 405nm, 488nm, 561nm, and 633/638nm, allowing users to first optimize panel(s) on the LSR II or Fortessa, and subsequently transfer to the sorter.

The BD Aria II is able to sort up to 4 populations simultaneously; the cuvette flow cell design eliminates the need for daily alignment; the cuvette also allows better detection of the photons emitted from the fluorochromes; finally, the Aria uses the same software, Diva, as the analyzers for operation, which makes the training for self-operation easier.

The BD Influx can sort up to six populations simultaneously, with a jet-in-air flow cell design reducing stress on the cells, and can sort single cell into both 96- and 384-well format.

These two complementary systems enable studies with maximum resolution and high speed, with the gentleness provided by Influx facilitating high viability rates needed for transplant and RNAseq studies.

Standards, Rigor, and Reproducibility

Flow Cytometry: Daily laser alignment and fluidics maintenance of analytical equipment is performed. Expert consultation for all prospective users assessing the needs of their research programs and providing detailed information on the FCSR instrumentation, capabilities, and services. Detailed consultation and troubleshooting on sample preparation, staining protocol optimization, and multi-color panels design with PhD-level staff scientists.             Training in flow data analysis and interpretation focused primarily on complex flow data analysis, including high-dimensional data analysis (15-30 parameters), using FCS Express. Training of users on best practices in flow cytometry in support of research rigor and reproducibility.

Flow Cytometry Cell Sorting Service: Cell sorter operation requires constant monitoring and expertise decision throughout the process. To ensure quality, all sorters are operated by well-trained and dedicated staff. The responsibility of the users is to prepare the single cell suspension according to our protocols while FCSR staff will ensure that laser intensity, stream stability, cooling system, biosafety, drop delay, sort stream trajectory, calibration of the tube/plate position, and sort processes are all up to the specifications set by the manufacturer and the FCSR. All the sorters go through systemic sterilization with 70% ethanol every 2 weeks, allowing the continued culture of the cells after sorting.

Flow Data Analysis and Troubleshooting: Training is available for flow analysis packages, including FlowJo and FCS Express. Staff go through the first few sets of data with users to ensure correct instrument settings, proper compensation for spillovers, and adequate controls. FCSR staff establish the workflow for data analysis including cleaning up data, setting the gates, generating statistics, and data presentation. Given ever-evolving flow cytometry field, the FCSR continually evaluates other algorithms (e.g., Cytobank, Modfit, and R-programming).