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Cell Cytometry and Sorting Core

The Cell Cytometry and Sorting Core provides the latest technology for cutting-edge flow cytometry research. The core facilities assist researchers in simultaneous, rapid examination of diverse individual cell types within a population of cells suspended in fluid. The Cell Cytometry and Sorting core is directed by Anda Vlad, MD PhD, Associate Professor, and managed by Aaron Siegel.

The facility is located on the 4th floor of MWRI, and it employs two instruments — BD LSRII and BD FACSARIA Fusion — for analytical and sorting experiments, respectively.

The 3 laser LSRII instrument is equipped with blue (488 nm), red (633 nm), and violet (405 nm) lasers, allowing for simultaneous analysis of up to eight colors. This laser configuration and the octagon detector array with user-interchangeable reflective dichroic and optically matched bandpass filters allow for multicolor experiments to be conducted with more flexibility and improved ease of use. The LSRII is attached to an intranet-connected dual monitor workstation and a color printer, and is operated by FACSDiva software.

For sorting requirements, the Cell Cytometry and Sorting Core contains a FACSARIA Fusion set in a Biohazard Baker laminar hood for BSL2+ work. The ARIA Fusion features 5 lasers, including (488nm) blue, (561nm) yellow-green, (640nm) red, and (405nm) violet laser capable of simultaneously analyzing up to 11 fluorochromes. The instrument is also equipped with a (355nm) UV laser which can be used as an alternative to violet. Three different nozzles (70, 85, 100 microns) may be utilized for varying cell sizes. Samples may be sorted into 2 tubes (2-way sort) or 4 tubes (4-way sort), or sorted into 6, 24, 48, 96, or 384-well plates. An online scheduler is available to users for reserving instrument time for flow cytometry research.

Core Services

Aaron Siegel maintains and runs the instrument. He helps new users identify proper fluorochrome combinations; provides general protocols, if needed; runs samples and exports data to the server; provides users with printouts of analyzed data; provides guidance with statistical analysis; trains investigators who intend to become independent users; and provides and maintains scheduling.

Fee Structure:

Contact Aaron Siegel for current pricing fee.

User Options (LSRII)

  1. Assisted user: Researcher discusses setup and worksheet parameters with
    FACSDiva on LSRII with Joan for each experiment.
  2. Independent user: Researcher is familiar with FACSDiva, has set up experiment with Joan, and independently runs the instrument during work hours.
  3. Power user: Researcher is proficient with FACSDiva and LSRII flow cytometry, understands electronics, fluidics, start up, shut down, and has basic trouble-shooting experience. May work during off hours and weekends

Scheduling Time

Scheduling time on the LSRII:

  1. Scheduling is restricted to weekdays between 8 a.m. and 6 p.m. Only independent users can use the Cell Cytometry and Sorting Core after working hours. Instructions on how to become an independent user are listed below.
  2. Users are kindly asked to reserve no more than 2 hours at a time. If needed, longer experiments can be scheduled after consultation with Aaron.
  3. MWR LSR2Flow Shared Outlook calendar needs to be used for scheduling. When signing up, the following information needs to be included in the subject box: Name–Lab / Phone# / Grant#
  4. Cancellations will be honored up to one hour prior to your appointment.*

*Subject to change with heavy schedule bookings.

Scheduling time on the FACSARIA Fusion sorter:

  1. The MWRFACSsorter Shared Outlook calendar can be used for scheduling.
  2. Preferably, please contact Aaron via email (siegela@mwri.magee.edu) or phone (412-641-1796) at least 5 days in advance to ensure sort availability so that new accommodations can be made.
  3. Please keep in mind that prep and sort may take up to 3 hours.

Checklist for First-Time Core Users

  1. Contact Aaron Siegel to obtain username and password.
  2. Design experimental layout, including: sample tube order, corresponding markers and fluorophores used, and controls (antibody isotype controls, compensation controls).
  3. Confirm experimental layout with Aaron.
  4. Reserve time (see online scheduling directions below).
  5. Stain cells, perform the analysis, and become familiar with the FACSDiva software.
  6. Retrieve data from the server and perform further analysis, if needed.

Training session availability:

To become an independent user on the LSRII of the Cell Cytometry and Sorting Core, you must contact Aaron Siegel for training.

Additional Resources

New to flow? Not sure how this technology can help your research?

Principles of flow cytometry:

Cells (or any other particulate materials) are pre-labeled with fluorescent dye(s) in suspending medium and then passed through a narrow nozzle so that each cell (particle) is in a small droplet. Single cells are passed through a beam of laser light. Lasers excite the fluorescent dyes and the analytical measurements are based on how the dye reacts to the laser (emission spectra). Quantification of the dye(s) on the cell are determined. Cells may be physically separated in the sorter instrument as the drop containing the cell of interest has an electrical charge applied; as it flows by high-voltage charged plates, cells are thereby attracted or repelled and sorted into collection vessel(s).

Examples of various analyses possible with flow:

  1. Multifluorochrome analysis: Ability to simultaneously measure multiple
    parameters on a cell; mostly used to identify cell subtypes from blood or tissues (processed to single cell suspension by enzymatic digestion)
  2. Cell cycle analysis: Measurement of the DNA content of cells; information about the cell cycle, the effect on the cell cycle of added stimuli (e.g., transfected genes or drug treatment)
  3. Apoptosis analysis: Analysis of apoptotic events using markers such as annexins, caspases, cyclins, cyclin-dependent kinases, signaling molecules, and DNA fragmentation markers
  4. Functional analysis: As long as the relevant functional aspect of the cell can be tagged with a detectable marker, flow cytometry can be used to assess functionality, such as (a) Cytokine secretion assays, (b) Proliferation assays, (c) Calcium flux assays
  5. Stem cell analysis: Requires rare population identification based on some or all of the following (a) ALDH expression, (b) DNA content/proliferating capacity, (c) Phenotyping (multicolor analysis of multiple antigenic markers)

Links to protocols and related websites:

Location

Cell Cytometry and Sorting Core
Magee-Womens Research Institute
204 Craft Avenue, Lab B434B
Pittsburgh, PA 15213

Contact

Core Director

Anda Vlad, MD, PhD
P: 412 641 2985
E: avlad@mwri.magee.edu

Core Manager

Aaron Siegel
P: 412-641-1796
E: siegela@mwri.magee.edu