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Related Experiment Videos

A rapid mix flow cytometer with subsecond kinetic resolution

J P Nolan1, R G Posner, J C Martin

  • 1National Flow Cytometry Resource, Los Alamos National Laboratory, New Mexico 87545, USA.

Cytometry
|November 1, 1995
PubMed
Summary
This summary is machine-generated.

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This study presents a novel flow cytometry system that significantly reduces sample analysis time to under 1 second. This advancement enables precise kinetic measurements of rapid cellular events, improving mechanistic studies of cell function.

Area of Science:

  • Cellular and Molecular Biology
  • Biophysical Chemistry
  • Analytical Chemistry

Background:

  • Kinetic approaches are crucial for understanding cell function mechanisms.
  • Conventional flow cytometry has limitations in analyzing rapid cellular events due to long sample delivery times (10-20 seconds).

Purpose of the Study:

  • To adapt a stopped-flow rapid mixing device to a flow cytometer for sub-second mixing and measurement.
  • To overcome the temporal limitations of conventional flow cytometry for kinetic studies.

Main Methods:

  • Integration of a syringe-based stopped-flow rapid mixer with a commercial flow cytometer.
  • Modification of the mixer for slower sample delivery rates (1.8 microliters/s) compatible with flow cytometry.
  • Development of a custom nozzle holder with a fast-acting valve and low dead volume for a FACS stream-in-air nozzle.

Related Experiment Videos

  • Computer control of syringe motors, valves, and timing for precise kinetic measurements.
  • Utilizing light scatter gating for accurate particle selection and fluorescence measurement.

    • Main Results:

    • Achieved sample mixing and measurement in under 1 second, a significant improvement over conventional methods.
    • Demonstrated stable sample stream establishment within the sheath stream in under 1 second.
    • Obtained fluorescence measurements of microspheres with coefficients of variation of approximately 5%.
    • Enabled fluorescence measurements at times under 300 milliseconds using light scatter gating.
    • Validated efficient reagent mixing through iodide quenching of fluorescein isothiocyanate (FITC)-labeled microspheres.
    • Showcased precise control of reagent concentration and dilution through quantitative proportioning.
    • Successfully performed rapid kinetic measurements of intact cells, exemplified by FITC-formyl peptide binding to cell surface receptors.

    Conclusions:

    • The adapted flow cytometry system enables rapid kinetic measurements of cellular events with high precision.
    • This technology overcomes previous limitations, allowing for the study of faster biological processes.
    • The system offers precise control over reagent mixing and cell-sample interaction, enhancing mechanistic studies.