Flow Cytometry Resources

Microbiology 542: Immunology Lab
Microbiology 540: Immunology

University of Massachusetts, Amherst
provided by Eric Martz

Flow cytometry software and example data.

• The WinMDI program for analysis of listmode flow cytometry data files. (Available for PC/Windows, or Virtual PC/Softwindows on the Macintosh, but sorry, not for native Macintosh.) Generously provided by Joe Trotter of The Scripps Research Institute (TSRI). Here are detailed instructions on how to install WinMDI.
• PrintKey freeware for saving the image on the screen to a web-viewable GIF (Graphics Interchange Format) disk file, and and handing in your graphs. Here are detailed instructions on how to install PrintKey.
• Sample data file: 3-color human peripheral blood leukocytes (in Internet Explorer: right-click, Save Target As). Can you guess the specificity for each color?
• Sample data files: Calcium fluxes in human T cells (Jurkat cell line). In WinMDI, File, Preferences, Dotplot kinetics mode defaults, check Kinetics mode (sample entire file). Make a dotplot of Time vs. FL4-Ratio. In Format Dotplot, Draw options, check Kinetics mode. In each sample, the indicated reagent was added early, producing the change you can see in cytoplasmic calcium activity. Then manganese was added at the end to quench the signal from the calcium reporter (fura-2).
  • To download these files in Internet Explorer: right-click, Save Target As.
  • Calcium ionophore (control for maximum calcium influx)
  • Antibody to CD3 (OrthoKlone T3, OKT3)
  • Antibody to CD3 in the presence of a calcium-chelator (EGTA)

Immunology Lab 542

• What Is Flow Cytometry?
• Introduction to Flow Cytometry (Sorry, no graphics yet but the text is here.)
• Flow Cytometry Planning Assignment
• Antibodies Available
• Data Analysis Computer Lab Startup (paper handout)
• WinMDI: FC Data Analysis and Graphing
• Report Form: Corrected Fluorescence Intensities from Histograms

• Results: Dot Plots, Histograms, and Corrected Fluorescence Intensities
  • 2003 Control Fluorescence Intensities:
    Splenocytes FITC=2.5, PE=2.2 (file 026, 2nd Ab only) (File 025, autofluor., gave 2.5, 2.2)
    Thymocytes FITC=2.0, PE=2.0 (file 029, 2nd Ab only) (File 028, autofluor., gave 2.0, 1.9)
    "2nd Ab only" means FITC-goat anti-mouse-IgG not preceded by a "1st" mouse IgG antibody.
  • 2003 Graphics & CFI's by Students
    Updated 4:00 April 18.
    Previous updates: March 13, March 20, March 25, March 31, April 16.

  • 2002 Control Fluorescence Intensities:
    Splenocytes FITC=3.0, PE=3.1 (file 030) (File 029 gave 2.9, 3.0)
    Thymocytes FITC=2.2, PE=2.6 (file 026) (File 025 gave 2.2, 2.5)
  • 2002 Graphics & CFI's by Students

  • 2001 Control Fluorescence Intensities:
    Splenocytes FITC=3.6, PE=2.8 (file 002) (File 001 gave 3.5, 3.0)
    Thymocytes FITC=2.8, PE=2.3 (file 012) (File 011 gave 2.7, 2.3)
  • 2001 Graphics & CFI's by Students
  • 2001 Overview (Graphics by Eric)

  • 2000 Control Fluorescence Intensities:
    Splenocytes FITC=3.3, PE=1.7 (file 016)
    Thymocytes FITC=2.7, PE=1.5 (file 006)
  • 2000 Graphics & CFI's by Students
  • 2000 Overview (Graphics by Eric)

  • 1999 Control Files by Eric
  • 1999 Graphics & CFI's by Students
  • 1999 Overview (Graphics by Eric)

  • 1997 Overview (Graphics by Eric)

• Raw data (mouse mononuclear cells)
  If clicking on a data file doesn't automatically download it:
  • In Netscape: Shift-click.
  • In Internet Explorer: Right-click, then Save Target As.

  • 2003, keys to data files
  • 2003, splenocytes & thymocytes

  • 2002, keys to data files
  • 2002, splenocytes & thymocytes

  • 2001, keys to data files
  • 2001, splenocytes & thymocytes

  • 2000, keys to data files
  • 2000, splenocytes & thymocytes

  • 1999, keys to data files
  • 1999, splenocytes & thymocytes

  • 1997, keys to data files
  • 1997, splenocytes & thymocytes

  • 1995, key to data files
  • 1995, splenocytes
  • 1995, thymocytes

UMass Flow Cytometry Facility