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Related Experiment Videos

Discriminating between damaged and intact cells in fixed flow cytometric samples.

L W Terstappen1, V O Shah, M P Conrad

  • 1Becton Dickinson Monoclonal Center, Inc., Mountain View, California 94043.

Cytometry
|September 1, 1988
PubMed
Summary

A novel nucleic acid stain, LDS-751, effectively distinguishes intact from damaged cells in flow cytometry after paraformaldehyde fixation. This method enhances immunofluorescence accuracy by enabling clear cell population identification.

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Area of Science:

  • Cell biology
  • Biotechnology
  • Analytical chemistry

Background:

  • Flow cytometry is crucial for cell analysis.
  • Distinguishing intact from damaged cells is vital for accurate results.
  • Paraformaldehyde fixation is common but can complicate cell discrimination.

Purpose of the Study:

  • To evaluate LDS-751 as a vital stain for discriminating cell integrity in fixed samples.
  • To assess the compatibility of LDS-751 with multi-color immunofluorescence.
  • To improve the accuracy of flow cytometric analysis.

Main Methods:

  • Utilized LDS-751, a nucleic acid stain, for cell analysis via flow cytometry.
  • Fixed cell samples with paraformaldehyde.
  • Correlated LDS-751 fluorescence with cell integrity (intact vs. damaged) and identified cell types (erythrocytes, platelets).

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  • Confirmed findings using light microscopy and FDA staining.
  • Integrated LDS-751 with two-color immunofluorescence.

    • Main Results:

    • Identified three distinct cell populations based on LDS-751 fluorescence intensity in fixed cells.
    • Erythrocytes and platelets showed low/no staining; intact cells had intermediate staining; damaged cells exhibited high staining.
    • Agglutinated cells were identifiable by increased LDS-751 signal.
    • LDS-751's spectral properties allowed combined use with two-color immunofluorescence.
    • Demonstrated improved accuracy in immunofluorescence measurements.

    Conclusions:

    • LDS-751 is a reliable vital stain for differentiating intact, damaged, and other cell types in fixed samples using flow cytometry.
    • The dye facilitates accurate cell discrimination, even in the presence of agglutination.
    • Combining LDS-751 with immunofluorescence enhances multi-parameter analysis of fixed cells.