The Immunology Core Facility provides state-of-the-art equipment and research support capability in the areas of flow cytometry and cell sorting.

Flow Cytometry 

Flow cytometry is a powerful, high-throughput technique used to analyze cells or particles based on physical and fluorescent properties. Typical applications of flow cytometry analysis include phenotyping of immune cell populations, assessing expression levels of surface and intracellular antigens, signal transduction events, cell cycle progression, apoptosis, and quantification of cytokine levels through cytokine bead arrays, and bacterial agglutination. We maintain two legacy instruments, and two high-parameter instruments in the immunology core.  

BD FACSCalibur™

The BD FACSCalibur™ is a legacy analyzer equipped with a 488nm blue laser, 633nm red diode laser, and four fluorescent detectors. The FACSCalibur™ is a workhorse in the core for bacterial agglutination studies. Configuration: 

• BLUE 488nm LASER
  • 530/30 – FITC, GFP, Alexa 488
  • 585/42 – PE, PI
  • 661/16 – PerCP, 7AAD
• RED 633 LASER
  • 670 – APC, Alexa 647

BD FACSAriaII™

The BD FACSAriaII™ is a legacy sorter equipped with three air-cooled lasers (488nm, 633nm, 407nm) capable of detecting up to nine fluorescent parameters and two scatter parameters. The fixed alignment cuvette flow cell allows for superior fluorescence sensitivity. The instrument is capable of four-way sorting at a maximum rate of 25,000 particles per second, simultaneously. An Automatic Cell Deposition Unit (ACDU) can sort a predefined number of cells into individual wells of 96 well or 24 well plates. Configuration:

• BLUE 488nm LASER
  • 780/60 – PE-Cy7
  • 710/50 – PerCP-Cy5.5, PE-Cy5.5, PI
  • 610/20 – PE-Texas Red
  • 575/26 – PE
  • 530/30 – FITC, GFP
• RED 633nm LASER
  • 780/60 – APC-Cy7, Alexa 780
  • 670/30 – APC, Alexa 647
• VIOLET 405nm LASER
  • 525/50 – BV510
  • 450/50 –BV421, Pacific Blue, DAPI

BD FACSymphony™ A3

The BD FACSymphony A3 is a high-parameter analyzer equipped with four lasers (488nm, 561nm, 640nm, 405nm) capable of detecting up to 16 fluorescent parameters and two scatter parameters. The enhanced capabilities of the A3 analyzer permit the use of new dyes with improved brightness and spillover characteristics, allowing for flexible panel design and better resolution of rare cell subsets. 

• BLUE 488nm LASER
  • 670/40 – PerCP, PerCP-Cy5.5, RB705
  • 530/30 – FITC, GFP, Alexa 488, BB515
• YELLOW-GREEN 561nm LASER
  • 780/60 – PE-Cy7
  • 710/50 – PE-Cy5.5
  • 670/30 – PE-Cy5, 7AAD
  • 610/20 – PE-Texas Red, PE-CF594, Alexa 568, mCherry, mScarlet
  • 586/15 – PE
• RED 640nm LASER
  • 780/60 – APC-Cy7, Alexa 750
  • 730/45 – Alexa 700
  • 670/30 – APC, Alexa 647
• VIOLET 405nm LASER
  • 780/60 – BV 786
  • 710/50 – BV711
  • 660/20 – BV650
  • 610/20 – BV605
  • 525/50 – BV510, Pacific Green, AmCyan
  • 431/28 – BV421, Pacific Blue, DAPI

BD FACSymphony™ S6

The BD FACSymphony S6 is a high-parameter sorter equipped with five lasers (488nm, 561nm, 640nm, 405nm, 349nm) capable of detecting up to 23 fluorescent parameters and two scatter parameters. The enhanced capabilities of the S6 sorter permits the use of new dyes with improved brightness and spillover characteristics, allowing for flexible panel design and better resolution of rare cell subsets. In addition, the S6 is capable of 6-way sorting at a maximum rate of 25,000 events per second, simultaneously. An Automatic Cell Deposition Unit (ACDU) can sort a predefined number of cells into individual wells of 24, 96, 384, and 1536 well plates. Configuration:

• BLUE 488nm LASER
  • 780/60 – BB791
  • 710/50 – BB700
  • 670/30 – PerCP, PerCP-Cy505, RB705
  • 515/20 - FITC, GFP, Alexa 488, BB515
• YELLOW-GREEN 461nm LASER
  • 780/60 – PE-Cy7
  • 670/30– PE-Cy4, 7AAD
  • 610/20 – PE-Texas Red, PE-CF594, Alexa 568, mCherry, mScarlet
  • 586/15 - PE
• RED 640nm LASER
  • 780/60 – APC-Cy7
  • 730/45- Alexa 700
  • 670/30 – APC, Alexa 647
• VIOLET 405nm LASER
  • 780/60 – BV786
  • 710/50 – BV711
  • 670/30 – BV650
  • 610/20 – BV605
  • 470/14 – BV480, DAPI
  • 431/28 – BV421, Pacific Blue
• ULTRAVIOLET 349nm LASER
  • 820/60 – BUV805
  • 740/35 – BUV737
  • 670/30 – BUV661
  • 586/15 – BUV563
  • 515/30 – BUV496
  • 379/28 – BUV396

Sample Requirements

Each sample requires a single cell suspension in a non-protein buffer, at a concentration of 0.5 to 2.0 x 10⁶ cells/ml in a volume of 0.5 to 1.0ml in a 12x75 round bottom tube. Users also provide appropriate medium for cell collection after sorting. All samples must be filtered prior to sorting. All flow cytometry experiments require an unstained control, and single-color compensation controls for each fluorescent dye labeled antibody. 

Flow Cytometry Policies

• Safety: Samples exceeding BSL-2 cannot be analyzed or sorted in this facility unless the biohazards are inactivated through fixation.
• Making appointments: You need a lead time of at least 48 hours assisted use on an analyzer, and 7 days for cell sorting.
• Data and Analysis: Users are responsible for storing flow data. Please bring a USB thumb drive to copy data. Off-line computers are available for analysis. Please check with core staff for convenient times to learn data analysis.