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Related Experiment Videos

Cloning of human lymphocytes using limiting dilution.

A A Morley, K J Trainor, R S Seshadri

    Experimental Hematology
    |May 1, 1983
    PubMed
    Summary

    A new microwell cloning technique enables efficient lymphocyte culture. This method accurately enumerates clone-forming cells (CFCs) and isolates lymphocyte clones with high plating efficiency.

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    Area of Science:

    • Immunology
    • Cell Biology

    Background:

    • Lymphocyte cloning is crucial for immunological studies.
    • Existing methods for enumerating clone-forming cells (CFCs) can be inefficient.

    Purpose of the Study:

    • To develop a simple and efficient technique for cloning lymphocytes at limiting dilution.
    • To establish a reliable method for enumerating CFCs and isolating lymphocyte clones.

    Main Methods:

    • Utilized phytohaemagglutinin, irradiated cells, and interleukin-2-containing conditioned medium.
    • Employed microwell culture for limiting dilution cloning of lymphocytes.
    • Conducted quantitative considerations, mixing experiments, and replating experiments.

    Main Results:

    • Achieved a mean CFC frequency of 29.4% for fresh peripheral blood lymphocytes and 25.5% for continuously cultured lymphocytes.
    • Demonstrated that most positive microwells originated from single cells with high self-renewal capacity.
    • Observed sustained growth and high plating efficiency in microwell cultures.

    Conclusions:

    • The developed microwell technique is effective for cloning lymphocytes.
    • This method is suitable for enumerating clone-forming cells and isolating lymphocyte clones.
    • The technique offers high plating efficiency and sustained cell growth.

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