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The development of flow cytometry techniques began in 1934 with initial attempts by Andrew Moldavan, a bacteriologist who counted the cells in a flowing capillary system. Moldavan pumped cells through a capillary tube focused under a microscope for visualization. The invention of photometry allowed the measurement of differentially-stained cells, and Louis Kamentsky developed the first multiparameter flow cytometer in 1965 to identify and count the cancer cells in cervical tissue specimens.
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Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro
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Image-based flow cytometry technique to evaluate changes in granulocyte function in vitro.

Brian K McFarlin1, Adam S Venable2, Eric A Prado2

  • 1Applied Physiology Laboratory, University of North Texas; Brian.Mcfarlin@unt.edu.

Journal of Visualized Experiments : Jove
|January 16, 2015
PubMed
Summary
This summary is machine-generated.

This study introduces a novel assay to simultaneously measure granulocyte phagocytosis and oxidative burst, identifying four distinct activation phenotypes. This method offers a more comprehensive understanding of immune cell function in response to infection.

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Area of Science:

  • Immunology
  • Cellular Biology
  • Biotechnology

Background:

  • Granulocytes are crucial for innate immunity against bacterial and viral infections.
  • Existing methods for measuring granulocyte function are limited, often assessing only one function or providing a single activation percentage.
  • Current assays lack the ability to provide detailed insights into the complex interplay of granulocyte functions.

Purpose of the Study:

  • To develop and demonstrate a technique for the simultaneous measurement of granulocyte phagocytosis and oxidative burst.
  • To identify and characterize unique phenotypes of activated granulocytes based on combined functional assessments.
  • To evaluate the impact of incubation duration on granulocyte activation phenotypes.

Main Methods:

  • Simultaneous measurement of bacterial phagocytosis and oxidative burst in granulocytes.
  • Utilized an image-based flow cytometer with quantitative imaging (QI) capabilities and multiple lasers.
  • Assessed granulocyte phenotypes across serial time incubations (10, 20, 40 min) and a control on ice.

Main Results:

  • Identified four distinct granulocyte activation phenotypes: inactivated, low activation, moderate activation, and high activation.
  • High activation phenotype characterized by high phagocytosis, high oxidative burst, and co-localization of these functions.
  • Demonstrated that incubation time influences the redistribution of these activated granulocyte phenotypes.

Conclusions:

  • The developed assay provides a more comprehensive assessment of granulocyte function compared to existing methods.
  • Simultaneous measurement reveals distinct activation states, offering deeper insights into immune responses.
  • This technique has the potential to detect how treatments spatially affect granulocyte function over time.