The Immunology Core Facility provides state-of-the-art equipment and research support capability in the areas of Flow Cytometry and SPR (Surface Plasmon Resonance).
ProteOn™ XPR36 Surface Plasmon Resonance (SPR) System: The Bio-Rad ProteOn™ XPR36 protein interaction array system enables label-free quantitative analysis of biomolecular interactions in real time using surface plasmon resonance (SPR) technology. The interactions are monitored over time by flowing an analyte in a microfluidic channel over a ligand immobilized on a sensor chip and detecting the binding of the analyte to the ligand by measuring changes in the propagation of electromagnetic waves at the sensor surface. Advantages include 6 Injections in parallel, simultaneous 36 interactions and real-time parallel referencing.
Multiple sensor chip types for applications:
- GLC - Compact capacity amine coupling for protein-protein interactions
- GLM - Medium capacity amine coupling for protein-protein and protein-small molecule interactions
- GLH - High capacity amine coupling for protein-small molecule interactions
- NLC - NeutrAvidin for biotinylated molecule capture
- HTG - Compact capacity tris-NTA for histidine-tagged large molecule capture
- HTE - High capacity tris-NTA for histidine-tagged small molecule capture
- LCP - Used with the LCP Capturing Reagent Kit for liposome capture
Flow Cytometry: Flow cytometry can be used to assess the immunophenotype of cell types, specific cell receptor(s), ligand binding, expression of surface and intracellular antigens, signal transduction events such as calcium flux, cell cycle progression based on analysis of DNA and RNA, and apoptosis. The Sorter is a cytometer cable of collecting selected cell populations. There are two different flow cytometers in the immunology core.
Becton Dickinson FACSCalibur: A modular benchtop flow cytometer designed for applications that range from routine clinical work to advanced research. This system analyzes cells as they pass one at a time through a focused laser beam measuring several parameters, including: forward light scatter, side light scatter, several fluorescence parameters as well as pulse area width of any fluorescence parameter. Our core’s FACSCalibur is equipped with a second red diode laser offering additional flexibility in fluorochrome choice in multicolor research analysis. Lasers and detectors:
- BLUE LASER 488 nm. Detectors and intended dyes:
- 530/30 - FITC
- 585/42 - PE or PI
- 661/16 - PerCP or 7AAD
- RED LASER 633 nm. Detector and intended dyes:
- 670 – APC or Alexa Fluor 647
The Becton Dickinson FACSAria II Cell Sorter A bench top sorter that incorporates a fixed-alignment cuvette flow cell for superior fluorescence sensitivity. With three air-cooled lasers with wavelengths at 488 nm, 633 nm and 407 nm, it is capable of detecting up to 9 colors and 2 scatter parameters. This instrument is capable of 4-way sorting at maximum 70,000 particle/sec simultaneously. An Automatic Cell Deposition Unit (ACDU) sorts a predefined number of cells into individual wells of a 96 or 24 well plate or to slides. Lasers and detectors:
- BLUE LASER 488 nm. Detectors & intended dyes :
- 780/60 - PE-Cy7
- 695/40 - PerCP-Cy5.5 or PI
- 675/20 - PerCP
- 610/20 - PE-Texas Red
- 575/26 - PE or PI
- 585/42 - PE or PI
- 530/30 – FITC
- RED LASER 633 nm. Detectors & intended dyes
- 780/60 - APC-Cy7
- 660/20 – APC
- VIOLET LASER 407nm. Detectors & intended dyes:
- 530/30 - Alexa Fluor 430
- 450/40 - Cascade Blue, Pacific Blue, Hoechst, DAPI or Alexa Fluor 405
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 1.0 ml, in the flow sample tube (BD 12 x 75 mm Falcon 5ml round bottom polystyrene, REF 352054). Users provide appropriate medium for cell collection after sorting. All samples must be filtered prior to sorting. Flow tube filters (BD 12 x 75 mm Falcon 5ml round bottom polystyrene, REF 352235). All flow cytometry experiments require negative stain control, and 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 24 hours for FACSCalibur analyzer and 48 hours for FACS Aria II 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.