Flow Cytometry Shared Resource Services

Service Profile

• Aseptic sorting of living cell populations (up to 4 simultaneous collection tubes)
• Direct cloning into microtiter tissue culture plates or onto microscope slides
• Isolation of rare cell subpopulations or large number of cells
• Quantitative fluorescence analysis (up to 9 colors)
• Consulting (design of experiments; analysis or conversion of data)
• Typical applications include:
  • Viability and toxicity assays
  • Apoptosis assays (TUNEL, sub-G1, caspases, Annexin V, mitochondrial membrane potential, etc)
  • Calcium Flux
  • Intracellular pH
  • Degranulation
  • Oxidative Burst
  • Phagocytosis
  • Reticulocytes
  • Platelets
  • Bead-based assays

Service Details

1. Analytical Flow Cytometry Utilizes a Becton Dickinson FACSCalibur, a 4-color, dual-laser benchtop flow cytometer. Included is the CellQuest software for list-mode data recording and analysis. Data recording and analysis can be performed with the FACS Aria, a triple-laser, multiparameter flow cytometer using up to 11-color fluorescence signals, along with forward and side-angle light scatter.
2. Cell Sorting In addition to analytical experiments, the FACSAria can perform aseptic sorting of living cell subpopulations utilizing various criteria, such as intrinsic cell properties, e.g. DNA content, Green Fluorescent Protein (GFP), or extrinsic properties, such as molecular markers identified by fluorescently-labelled monoclonal antibodies.
3. Automated Magnetic Cell Sorting (autoMACS) Used for convenient isolation of rare cell subpopulations or large numbers cells, up to 4 x 109. Its use of magnetic particles for cell separation is both useful and efficient, and the cells can then go straight to FACS analysis or further sorting. Manufacturer's website: autoMACS
4. Consultation In addition to education about the functionality and capabilities of the Shared Resource, technical support personnel also advise in the design of experiments and assist investigators in the initiation of pilot projects, provide limited amounts of specific reagents, and contribute detailed consultation in analysis of data.

Examples

• Toxicity and viability assays (PI, NK assays, CTL, etc.)
• Many apoptosis assays (DNA content, TUNEL, SBIP, Annexin-V, caspases , mitochondrial membrane potential, etc.)
• Quantification of cells expressing specific surface, cytoplasmic, secreted, or nuclear antigens
• Proliferation assays (BrdU, PKH-26, CFDA-SE etc.)
• Cell Cycle Analysis (by quantification of DNA or incorporation of BrdU)
• Separation of living cell populations expressing marker genes for specific cell lineages (GFP, FACS-gal, NGFR, etc.)
• Cloning of cells modified by introduction of novel genes for therapeutic purposes (CloneCyt Plus)
• Calcium Flux (Fluo-4, Fluo-3/Fura Red, Indo-1)
• Membrane Potential
• Enzyme Activity
• Glutothione Determination (CellTracker, NDA, etc.)
• Multidrug Resistance (MDR) Assays
• Intracellular pH (SNARF-1)
• Phagocytosis (HDCF-BSA, Dihydrorhodamine 123, calcein, bacteria, yeast etc.)
• Oxidative Burst, Free Radicals & Reactive Oxygen Species
• Degranulation & Actin Polymerization
• Quantification of Reticulocytes
• Cytometric Bead Arrays
• Platelet Activation
• Aseptic sorting of any subpopulation of cells from suspension into tubes or microtiter culture plates or directly onto slides
• Index Cell Sorting: The well location is identified and recorded when a cell is sorted, making correlation of flow cytometry data (e.g., fluorescence intensity) with non-flow based assays more accurate.

Equipment

• BD FACSAria (High speed, 9-color, tripe-laser cell sorter)
  • Automated Cell Deposition Unit (ACDU)
  • 4-way sorting
• BD FACSAria II SORP (High speed, 13-color, 4-laser cell Sorter)
  • Automated Cell Deposition Unit (ACDU)
  • 4-way sorting
• BIO-RAD S3 (2-laser, 4-color cell sorter)
• BD FACSCalibur (2) - 4-color, non-sorting cytometers

Flow Cytometry Links

• Cytometry (Journal)
• International Society for Analytical Cytology (ISAC)
• The Scripps Research Institute Flow Cytometry Core 
• Molecular Probes
• Joe Trotter's Java Spectrum Viewer
• Purdue Cytometry E-Mail Archives
• University of Pittsburgh Medical Center Hillman Cancer Center