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Related Experiment Videos

Fluorescence-intensity multiplexing: simultaneous seven-marker, two-color immunophenotyping using flow cytometry.

Jolene A Bradford1, Gayle Buller, Michael Suter

  • 1Flow Cytometry Department, Molecular Probes, 29851 Willow Creek Road, Eugene, OR 97402, USA. jolene.bradford@probes.com

Cytometry. Part a : the Journal of the International Society for Analytical Cytology
|September 24, 2004
PubMed
Summary

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Zenon immunolabeling technology enables multiparameter flow cytometry by using fluorescence intensity to identify multiple targets per channel. This method allows for more sophisticated analysis on simpler flow cytometers.

Area of Science:

  • Immunology
  • Biotechnology
  • Cell Biology

Background:

  • Conventional flow cytometry requires a dedicated channel per antibody-fluorophore, limiting multiplexing capabilities.
  • Advanced flow cytometers with 10-15 channels have been developed to overcome these limitations.
  • Zenon immunolabeling technology offers a novel approach to multiplexing.

Purpose of the Study:

  • To develop and evaluate Zenon immunolabeling technology for fluorescence-intensity multiplexing in flow cytometry.
  • To enable simultaneous detection of multiple antigens per channel using a single fluorophore.
  • To demonstrate the applicability of this technique for lymphocyte immunophenotyping.

Main Methods:

  • Developed Zenon labeling reagent-antibody complexes with varying molar ratios.

Related Experiment Videos

  • Applied fluorescence-intensity multiplexing to label multiple antibodies with single fluorophores (Alexa Fluor 488 and R-PE).
  • Performed lymphocyte immunophenotyping on 38 clinical blood specimens using conventional and multiplexing analyses.

    • Main Results:

    • Lymphocyte immunophenotyping results from fluorescence-intensity multiplexing were comparable to conventional flow cytometric analysis.
    • Zenon labeling allowed simultaneous detection of seven markers using only two photomultiplier tubes (PMTs).
    • Distinguishable multivariate patterns were generated by labeling antibody sets with different Zenon reagents.

    Conclusions:

    • Zenon immunolabeling enables simultaneous evaluation of multiple antigens per fluorophore by varying labeling reagent ratios.
    • This technique allows for more cellular targets to be resolved than available detection channels.
    • Sophisticated multiparameter flow cytometry can be performed on less complex, 2- or 3-detection-channel instruments, resolving approximately 2-4 targets per channel.