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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
Summary
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.
- 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.