Flow Cytometry and Immunologic Analysis

Key Personnel

Peter Keng, Ph.D
Co-Scientific Director, URMC Flow Cytometry Core

Timothy Bushnell, Ph.D.
Co-Scientific and Technical Director URMC Flow Cytometry Core


Visit the Flow Core website

 

Mission Statement:

Our mission is to support and enrich research personnel and programs to maximize results and research dollars in an environment that welcomes the challenge of breaching previously unexplored avenues in polychromatic flow cytometry.

Core Services/Functions:

Services provided by the Core include:

  1. Immunofluorescent measurements (surface and intracellular markers, single and multi-parameters) for antigen and antibody distributions and cell signaling pathways.
  2. DNA, RNA, and nuclear antigens for cell cycle, apoptosis, and cell proliferation (BrdU) analysis.
  3. Sterile cell sorting and auto-cloning based on light scatter and fluorescent intensities (antibodies, DNA, RNA, GSH, GFP, etc.).
  4. Measurement of cellular functions such as Ca++ flux, redox status, NADP/NADPH, proliferation (CFSE), gene expressions (GFPs), FRET, etc. for analysis and cell sorting.
  5. Bulk cell separation by centrifugal elutriation for large quantities of host and malignant cells, and cells at different phases of the cell cycle.
  6. Imaging Cytometry
  7. Consultation and assistance in the development and implementation of new protocols and methodologies.
  8. Routine daily Quality Control and calibration check of all core instruments.

Instrumentation and Equipment:

Cell Sorting and Elutriation

  • BD FACS Vantage(Diva) 3-Laser (360 nm, 488 nm and 633 nm) and 6 fluorescent detectors. Currently located in 3-4100
  • BD FACSAria: 3-laser (405, 488 and 633 nm), 13 PMT detectors, for a total of 15 parameters.  Additional options include Aerosol management system (AMO; for BSL-2 containment), cooling option, and Automated Cell Deposition Unit (ACDU: for plate sorting).  Currently located in 1-11314
  • One Beckman JE-6 Elutriator system: Centrifuge system capable of cell separation based on density.  Currently located in 3-4100

Analytical Instrumentation

  • Two BD FACS Calibur Analyzers equipped with 488nm and 635 nm excitations and 4 fluorescent detectors.  Currently located in 3-4100 and 3-9655
  • BD FACS Canto:  3-laser (405 nm, 488 nm and 633 nm), 8 florescent detector digital system.  Currently located in 1-11314
  • BD FACS Canto:  2-laser (488 nm and 633 nm), 6 florescent detector digital system. Currently located at the Center for Cardiovascular Research, on Bailey road
  • BD LSR-II: 3-laser (405 nm, 488 nm and 633 nm), 11 fluorescent detectors  Currently located in 2-9905
  • BD LSR-II: 4-laser (405 nm, 488 nm, 532 nm and 633 nm), 18 fluorescent detectors. Currently located in 2-9905
  • Amnis ImageStream:  Imaging cytometer with three lasers capable of collecting 6 multispectral images.  Currently located in 1-11314
  • BioRad BioPlex: Bead micro-array flow cytometer.  Currently located in 2-9905

Flow Cytometry Oversight Committee:

Craig Jordan (co-chair)
Tim Mosmann (co-chair)
Steve Dewhurst, Senior Associate Dean
Steve Goldman
Michael McCabe
Richard Phipps
Inaki Sanz
David Topham
Sally Quataert

Cell Sorting Core

Director

Peter Keng, Ph.D.


Visit the Cell Sorting Core website

 

Mission Statement

The Cell Sorting Core at the University of Rochester Medical Center provides state-of-the-art instrumentation, education and expert technical assistance to investigators for the successful performance of flow cytometry based studies. The goal of the Core is both to support current research applications and to continuously extend the repertoire of flow cytometric methods available to Cell Sorting Core users.

Statement of Core Services/Functions

The routine services provided by the Core include:

  1. Immunofluorescent measurements (surface and intracellular markers, single and multi-parameters) for antigen and antibody distributions.
  2. DNA, RNA, and nuclear antigens for cell cycle, apoptosis, and cell proliferation (BrdU) analysis.
  3. Sterile cell sorting and auto-cloning based on light scatter and fluorescent intensities (antibodies, DNA, RNA, GSH, GFP, etc.).
  4. Measurement of cellular functions such as Ca++ flux, redox status, NADP/NADPH, proliferation, gene expressions (GFPs), FRET, etc. for analysis and cell sorting.
  5. Bulk cell separation by centrifugal elutriation for large quantities of host and malignant cells, and cells at different phases of the cell cycle.
  6. Consultation and assistance in the development and implementation of new protocols and methodologies.

Specific Instrumentation/Equipment

  • One B-D FACS Vantage(Diva) Sorter equipped with 360 nm, 488 nm and 633 nm excitations and 6 fluorescent detectors.
  • Two B-D FACS Calibur Analyzers equipped with 488nm and 635 nm excitations and 4 fluorescent detectors.
  • One Beckman JE-6 Elutriator system.

 

Faculty Advisory Group

Craig Jordan (Chair)
Tom Gasiewicz
Jim Melvin
Edith Lord
Rick Phipps
Mike McCabe
Tim Bushnell
Sally Quataert

The Center for Pediatric Biomedical Research Flow Facility

Faculty Director

Craig Jordan, Ph.D.


Visit the The Center for Pediatric Biomedical Research Flow Facility website

 

Technical Director

Tim Bushnell, Ph.D.

Mission Statement

The goal and mission of the Center for Pediatric Biomedical Research flow facility is to develop, validate and implement polychromatic flow cytometric protocols to support and enrich the research programs of CPBR faculty. This flow facility will focus on the development of experimental and unique protocols to breach previously unexplored avenues of experimentation with flow cytometry.

Core Services/Functions

The CPBR flow lab core operation currently provides the following services.  

  • High-speed cell sorting using the FACSAria cell sorter. 
  • Imaging Cytometry using the Amnis ImageStream
  • Consultation on polychromatic flow cytometric experimental design
  • Data analysis support

Instrumentation/Equipment

  • BD FACSAria: 3-laser (405, 488 and 633 nm), 13 PMT detectors, for a total of 15 parameters.  Additional options include Aerosol management system (AMO; for BSL-2 containment), cooling option, and Automated Cell Deposition Unit (ACDU: for plate sorting)
  • Amnis ImageStream:  A single 488 nm Laser capable of collecting 6 multispectral images.  Instrument is currently on a non-capital lease agreement.
  • PC workstation for data analysis, equipped with IDEAS software.
  • MAC workstation for data analysis, equipped with Flowjo software.

Faculty User group

The CPBR FACSAria is a dedicated instrument, however, approximately 50% time is available to the URSMD scientific community.  There is no faculty user group for this instrument, but major users include Craig Jordan, Michael Becker, James Palis, and Inaki Sanz.

Amnis ImageStream is currently supported by the Cancer Center and is available to the URSMD community as a core instrument.  Major users of the Amnis ImageStream include Craig Jordan, Jim Palis, Rick Phipps, Steve Goldman and Inaki Sanz.

Human Immunology Center Laboratory

Director

Sally Quataert, Ph.D.


Visit the Human Immunology Center Laboratory website

 

Mission Statement

The Human Immunology Center (HIC) and its’ core laboratory were established to acquire, refine and develop expertise in cutting-edge techniques and to support applications in Human Immunology research.  The HIC serves to enhance multi-disciplinary research initiatives catalyzing key clinical and basic immunology research in vaccines, HIV/AIDS, autoimmunity, allergy/asthma, transplantation as well as cancer immunology.  The Center’s core lab provides assistance and expertise in immunological method development, standardization and validation through individual and group training programs

Services

The HIC Core lab offers researchers access to multicolor flow cytometry analysis and Luminex multiplexed microbead array technology.  Researchers receive training on multiplexed cell and bead array techniques as well as instrument operation and are granted access to HIC equipment. The BD LSRII flow cytometer is available with on-line booking to both human and animal researchers using more than 4-color flow applications.  The Director is available for consultation on clinical and translational projects that involve the use of human samples or may require compliance with Good Clinical Laboratory Practice Standards (GCLP) and/or Good Laboratory Practice (GLP) regulations.

Instrumentation/Equipment

  • BD LSR-II analytical flow cytometer. This is a 3-laser, 11-color 13-parameter instrument with secure data servor and back-up system.
  • BioRad BioPlex Microbead Array (Luminex 200) Flow cytometer system with Analysis Software

Faculty User Group

The HIC Core Lab is overseen by the Human Immunology Center Executive Committee.