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Related Experiment Videos

A new microcellular cytotoxicity test based on calcein AM release

X M Wang1, P I Terasaki, G W Rankin

  • 1UCLA Tissue Typing Laboratory, Department of Surgery, UCLA School of Medicine 90024.

Human Immunology
|August 1, 1993
PubMed
Summary

This study introduces a new microtest for cell-mediated immunity, significantly reducing cell requirements and enabling rapid, accurate detection of immune cells like NK cells, LAKs, and CTLs.

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Area of Science:

  • Immunology
  • Cell Biology
  • Biotechnology

Background:

  • Cell-mediated immunity is crucial for host defense.
  • Existing assays for cell-mediated immunity can be resource-intensive.
  • There is a need for more efficient and sensitive methods to study immune cell function.

Purpose of the Study:

  • To develop and validate a novel microtest for assessing cell-mediated immunity.
  • To reduce the number of cells required for immune assays.
  • To enable rapid and accurate quantification of immune cell activity.

Main Methods:

  • Utilized the Tarasaki tray and calcein AM vital dye for live cell staining.
  • Employed a fluorimeter for rapid (1 second/test) result acquisition.
  • Used ethidium bromide as a counterstain to confirm dead cells.

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  • Validated assay sensitivity and linearity with target cell dilutions.

    • Main Results:

    • Reduced target cell requirement to 500 cells/test and effector cells tenfold.
    • Demonstrated low spontaneous leakage rate (<15% in 4 hours at 37°C) for calcein AM.
    • Successfully detected Natural Killer (NK) cells, Lymphokine-Activated Killer (LAK) cells, and Cytotoxic T Lymphocytes (CTLs).
    • Showed a significant positive correlation with established 51Cr-release assays.

    Conclusions:

    • The developed microtest is a sensitive and efficient tool for studying cell-mediated immunity.
    • This method allows for rapid quantitation of cell killing and kinetic analysis.
    • The microtest is suitable for a wide range of applications in immunological research.