INTEGRATING FLOW CYTOMETRY INTO

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					          INTEGRATING FLOW CYTOMETRY INTO


 THE UNDERGRADUATE CELL BIOLOGY CURRICULUM



      With funds from a Course Curriculum and Laboratory Improvement (CCLI)
grant from the National Science Foundation (DUE-0411095), Dennis McGee and
Steven Tammariello have integrated the technology of flow cytometry into our
undergraduate Cell Biology Curriculum.

GOALS: Revise and introduce new laboratory experiments using flow cytometry
       into our existing BIOL 427 Cell Biology Laboratory.

        Create a new in-depth course on Principles of Flow Cytometry (BIOL
        480M) which would fit well with our Cell Biology Curriculum.



The BD Biosciences FACSCalibur Flow Cytometer:

  Analytical flow cytometer capable of detecting 4 colors
  Standard Argon-ion laser (488nm) and a second Red Diode laser (635nm)
  Sort Sensor Unit for cell sorting
  FACSLoader for automated sample handling
Flow Cytometry Laboratory Experiments added to the BIOL 427
Cell Biology Laboratory:

An Introduction to Flow Cytometry and Cell Cycle Analysis

       In this new experiment, the students
are introduced to the workings of the flow
cytometer by determining the effect of
insulin deprivation on cell cycle in cultured
rat IEC-6 intestinal epithelial cells. The cells
are then harvested, fixed and stained with
propidium iodide for cell cycle analysis
using the flow cytometer. As expected,
insulin-deprived cells showed a lower
percentage of cells in S or G2/M phases of
cell cycle.




Lymphoid Cells - Immunofluorescent Staining for Surface Proteins

      The original laboratory experiment
involved immunofluorescent staining of
mouse spleen cells for B lymphocytes using
an FITC-conjugated anti-mouse Ig antibody
and counting stained cells using a fluorescent
microscope. Students now use the
FACSCalibur flow cytometer to rapidly count
10,000 spleen cells per sample and determine
the percentage of stained B lymphocytes.



Independent Laboratory Research Projects

      The Cell Biology Laboratory course ends with student groups designing
and performing an independent research project using techniques learned in
the lab. They then present their findings to the other students in the form of a
Poster Session. Examples of Independent Projects performed by students using
flow cytometry include:

   Percentages of B cells vary between lymphoid tissues in a Swiss-Webster
      mouse
   Concanavalin A induces mouse spleen cells to progress into cell cycle
BIOL 480M Principles of Flow Cytometry:

       This course was designed to give students a more intense introduction
into the principles and techniques of flow cytometry. The laboratory
experiments included:

   - Lymphoid Cells - Differential White Blood Cell Counts
      (As an introduction to lymphocytes)

   - Quality Control with CaliBRITE Fluorescent Beads
   - Optimization and Compensation Using CaliBRITE Beads
      (These two are an introduction to the cytometer)

   - Lymphoid Cells - One Color Immunofluorescent staining of B lymphocytes
   - Cell Quest Pro Software

   - Multi-Color Immunofluorescent Staining of Spleen Cells for T Lymphocytes
      (Phycoerythrin-anti-mouse CD3 and FITC-anti-mouse CD8)

   - Preparation and Staining of Cancer and Non-Cancer Cells for Cell Cycle
      Analysis
   - Cell Cycle Analysis Using the ModFit LT Program
      (Cell cycle analysis of mouse spleen cells, non-transformed rat IEC-6
      intestinal epithelial cells and Caco-2 human colon carcinoma cells)

   - Independent Laboratory Research Project
     As in the Cell Biology Laboratory, the course ended with the student
groups performing an independent laboratory research project. Examples of
some projects included:

   LPS stimulation induces B Cells
      to enter cell cycle

   Cell cycle analysis of EGF and
      insulin stimulated cells

   Vybrant DyeCycle stain as a
      substitute for propidium
      iodide stain in cell cycle
      analysis.




We would be happy to discuss our experiences and findings with educators who
would like to try these experiments in their own curriculum, or provide copies
of any of our above experiments.

Please contact:

Dr. Dennis McGee
Department of Biological Sciences
Binghamton University (SUNY)
Binghamton, NY 13902-6000
Email: dmcgee@binghamton.edu