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Related Experiment Videos

Molecular calibration in flow cytometry with sub-attogram detection limit.

J V Watson, M J Walport

    Journal of Immunological Methods
    |November 6, 1986
    PubMed
    Summary

    Researchers calibrated flow cytometry using a novel method with a monoclonal antibody to complement receptor type 1 (CR1). This allows precise quantification of CR1 molecules on human granulocytes, improving cell analysis accuracy.

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    Clinical and experimental immunology·2003

    Area of Science:

    • Immunology
    • Cell Biology
    • Biotechnology

    Background:

    • Flow cytometry typically quantifies cellular targets using arbitrary fluorescence units.
    • Accurate quantification of cell surface molecules like complement receptor type 1 (CR1) is crucial for understanding cellular function and disease states.
    • Standardization of flow cytometry measurements is needed for reliable and reproducible results.

    Purpose of the Study:

    • To develop a method for calibrating flow cytometers using absolute numbers of molecules per cell.
    • To determine the precise number of CR1 molecules on normal human granulocytes.
    • To establish the detection limit and resolution of the calibrated flow cytometer for CR1.

    Main Methods:

    • A double-labelled monoclonal antibody to CR1 (with 125iodine and fluorescein isothiocyanate - FITC) was used for parallel radioimmune assay (RIA) and flow cytometry.

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  • Serial dilutions of labelled antibody were employed to correlate radioactivity counts with fluorescence intensity.
  • The flow cytometer was calibrated to report results in molecules per cell, not arbitrary fluorescence units.

    • Main Results:

    • A linear correlation was established between radioactivity and fluorescence for 700-25,000 labelled antibody molecules per cell.
    • Normal human granulocytes possess a mean of 25,000 CR1 molecules per cell (standard deviation 4775).
    • The flow cytometer achieved a detection limit of 730 CR1 molecules per cell and a resolution of 44 CR1 molecules per digitization step.

    Conclusions:

    • The developed method enables accurate calibration of flow cytometers in terms of absolute molecular counts per cell.
    • This technique provides a more precise quantification of CR1 expression on human granulocytes.
    • The findings advance the standardization of flow cytometry for quantitative cell surface marker analysis.