The Flow Cytometry Research Core

The Flow Cytometry Core provides the latest technology for cutting-edge flow cytometry research. The core facilities assist researchers in simultaneous, rapid examination and quantization of diverse individual cell types within a population of cells suspended in fluid. The Flow Cytometry research core is directed by Anda Vlad, MD, PhD, an immunologist, and managed by a fully trained technician, Joan Brozick. The facility is located on the 4th floor of MWRI, and is equipped with a new, 3-laser LSR II instrument (BD Biosciences). The blue (488 nm), red (633 nm), and violet (405 nm) lasers allow simultaneous analysis of up to eight colors. This laser configuration and the octagon detector array with user-interchangeable reflective dichroics 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 to a color printer, and operated by FACSDiva software. Upon acquisition, flow data is automatically transferred to each user’s password-protected storage space on MWRI’s servers. An online scheduler is available to users for reserving instrument time for flow cytometry research.

Location:

Magee-Womens Research Institute
204 Craft Ave., Lab B432
Pittsburgh, PA 15213

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) and then passed, in suspending medium, 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 measurements are based on how the dye reacts to the laser (emission spectra).

Examples of various analyses possible with flow:
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:
Contact Us:

Joan Brozick or 412-641-6106
Anda Vlad MD, PhD

Flow Cytometry Core Services and Fees

Flow Cytometry Research Services offered:

Joan Brozick maintains and runs the instrument. She 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 people who intend to become independent users, and; provides and maintains scheduling.

One free copy of the FACSDiva 6.1 software is available for checkout from Joan. Access to this shared copy (access key) may be limited, according to the number of user requests. Laboratories planning on utilizing the flow cytometry core frequently may consider purchasing their own software copy (contact Sue Hostler for more information).

Fee structure:

MWRI grant-managed scientist: $50/hour; Billed per-minute use: $0.83/minute
Non-MWRI grant-managed scientist: $65/hour; Billed per-minute use: $1.08/minute

Flow Cytometry Research Core Checklist for first-time users:
  1. Contact Joan Brozick 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 Joan.*
  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.
Scheduling time:
  1. Scheduling is restricted to weekdays between 9 a.m. and 6 p.m. Only independent users can use the flow cytometry research core after working hours. Instructions on how to become an independent user are listed below.
  2. Please do not reserve more than 2 hours at a time. If needed, longer experiments can be scheduled after consultation with Joan.
  3. PC users may utilize Outlook calendar for scheduling; Mac users may utilize Entourage.
  4. Directions for making an appointment:
  5. Make meeting under action
  6. Schedule available day/time
  7. Please include the following in the subject box: Name-Lab /Phone# /Grant#
  8. Cancellations will be honored one hour preceding your appointment.*

Subject to change with heavy schedule bookings.

User options:
  1. Assisted user: Researcher discusses set-up, runs options 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
Training session availability:

To become an independent user of the Flow Cytometry Research Core, you must contact Joan Brozick for training.