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Related Experiment Videos

Nine color eleven parameter immunophenotyping using three laser flow cytometry.

M Bigos1, N Baumgarth, G C Jager

  • 1Department of Genetics, Stanford University School of Medicine, California 94305-5318, USA. bigos@stanford.edu

Cytometry
|May 20, 1999
PubMed
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This study introduces a nine-color flow cytometry system for detailed cell analysis. Partial analog compensation improves data quality by reducing background noise, enabling the characterization of rare B cell populations.

Area of Science:

  • Immunology
  • Biotechnology
  • Analytical Chemistry

Background:

  • Describes a novel three-laser flow cytometer capable of analyzing nine distinct fluorescent parameters simultaneously.
  • Evaluates compensation strategies for multi-parameter flow cytometry data.
  • Presents an application for characterizing low-frequency murine B cell subpopulations.

Purpose of the Study:

  • To present a nine-color flow cytometry system.
  • To compare data quality between full software compensation and hybrid analog-software compensation.
  • To demonstrate the system's utility in identifying rare cell populations.

Main Methods:

  • Utilized an argon laser (488 nm), a pumped dye laser (595 nm), and a violet-enhanced krypton laser (407 nm).
  • Employed fluorochromes including FITC, PE, Cy5PE, Cy7PE, TR, APC, Cy7APC, CB, and CY.

Related Experiment Videos

  • Incorporated custom electronics and an extended optical bench for nine-parameter measurement plus scatter signals.

    • Main Results:

    • Partial analog compensation in hardware reduced background staining variations compared to full software compensation.
    • Successfully characterized novel B cell populations with frequencies below 1%.

    Conclusions:

    • Nine-color flow cytometry offers high information content for cell analysis.
    • Effective reagent-dye selection and compensation strategies are critical for accurate multi-parameter measurements.
    • The developed system is suitable for high-resolution immunophenotyping.