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Sensitive method for detecting viable cells seeded into bone marrow.

C P Reynolds, A T Black, J N Woody

    Cancer Research
    |November 1, 1986
    PubMed
    Summary
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    This study introduces a highly sensitive method for detecting viable tumor cells or lymphocytes in bone marrow. Using Hoechst 33342 (H342) DNA stain and fluorescence microscopy, researchers can identify single viable cells among millions.

    Area of Science:

    • Cell biology
    • Biotechnology
    • Medical diagnostics

    Background:

    • Accurate detection of viable cells in complex biological samples like bone marrow is crucial for diagnostics and research.
    • Existing methods may lack the sensitivity or specificity required for identifying rare cell populations.

    Purpose of the Study:

    • To develop and validate a highly sensitive method for detecting viable cells within mixed populations.
    • To assess the impact of the staining method on cell viability and susceptibility to lysis.

    Main Methods:

    • Staining viable tumor cell lines or normal lymphocytes with the supravital DNA stain Hoechst 33342 (H342).
    • Seeding stained cells into human bone marrow.
    • Utilizing an inverted fluorescence microscope for cell detection.

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  • Employing counterstaining with trypan blue to exclude nonviable cells.

    • Main Results:

    • The method allows for the detection of a single H342-stained viable cell in 1 million bone marrow cells.
    • Hoechst 33342 staining did not significantly alter cell viability.
    • Staining did not increase the sensitivity of cells to complement-mediated lysis.
    • Trypan blue counterstaining effectively quenched fluorescence in nonviable cells.

    Conclusions:

    • The described method provides highly sensitive detection of viable cells in mixed populations.
    • Hoechst 33342 is a suitable supravital stain for viable cell detection without compromising cell integrity or function.
    • This technique offers a valuable tool for identifying rare viable cells in biological samples.