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Related Concept Videos

Flow Cytometry01:23

Flow Cytometry

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The development of flow cytometry techniques began in 1934 with initial attempts by Andrew Moldavan, a bacteriologist who counted the cells in a flowing capillary system. Moldavan pumped cells through a capillary tube focused under a microscope for visualization. The invention of photometry allowed the measurement of differentially-stained cells, and Louis Kamentsky developed the first multiparameter flow cytometer in 1965 to identify and count the cancer cells in cervical tissue specimens.
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Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity
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Improvements in Flow Cytometry-Based Cytotoxicity Assay.

Xiaolong Wu1, Ying Zhang1, Yutao Li1

  • 1Department of Integrated Oncology, CIO Bonn, University Hospital Bonn, Bonn, D-53105, Germany.

Cytometry. Part a : the Journal of the International Society for Analytical Cytology
|October 17, 2020
PubMed
Summary

This study refines flow cytometry assays for cell-mediated cytotoxicity. It confirms no cross-staining with CFSE labeling and proposes fixed-time acquisition as a stable, efficient alternative to counting beads for accurate lysis evaluation.

Keywords:
CFSEHoechst 33258LAK cellsbeadscell-mediated cytotoxicity assayeffector-target conjugate formationflow cytometry

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Area of Science:

  • Immunology
  • Cell Biology
  • Biotechnology

Background:

  • Flow cytometry assays are preferred over radioactive methods for assessing cell-mediated cytotoxicity due to sensitivity and safety.
  • Concerns regarding dye leakage and cross-staining in flow cytometry cytotoxicity assays persist.
  • Optimizing assay parameters is crucial for accurate and efficient cytotoxicity measurements.

Purpose of the Study:

  • To investigate and resolve issues in flow cytometry-based cytotoxicity assays.
  • To evaluate the use of carboxyfluorescein diacetate succinimidyl ester (CFSE) for target cell labeling and Hoechst 33258 as a viability dye.
  • To establish an alternative standardization method to counting beads for improved assay efficiency and stability.

Main Methods:

  • Utilized CFSE to label target cells and Hoechst 33258 as a viability dye.
  • Assessed cross-staining between effector and target cells after co-culture.
  • Compared cytotoxicity overestimation when labeling effector versus target cells.
  • Investigated fixed-time sample acquisition as a standardization method.

Main Results:

  • Confirmed no cross-staining between CFSE-labeled target cells and effector cells after 4 hours of co-culture.
  • Demonstrated that labeling effector cells overestimates cytotoxicity due to viable target exclusion.
  • Showed that fixed-time acquisition provides stable and comparable results to counting beads for lysis evaluation.
  • Identified improved gating strategies using CFSE and forward scatter (FSC) for early apoptotic event detection.

Conclusions:

  • The study provides methods to enhance the accuracy and efficiency of flow cytometry-based cytotoxicity assays.
  • Labeling target cells and using fixed-time acquisition are recommended for reliable and cost-effective cytotoxicity assessment.
  • Optimized gating and standardization improve the overall performance and applicability of the assay.