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Related Concept Videos

Flow Cytometry01:23

Flow Cytometry

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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Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells
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Toward quantitative fluorescence measurements with multicolor flow cytometry.

Lili Wang1, Adolfas K Gaigalas, Gerald Marti

  • 1National Institute of Standards and Technology, Gaithersburg, MD 20899-8312, USA. lili.wang@nist.gov

Cytometry. Part a : the Journal of the International Society for Analytical Cytology
|January 1, 2008
PubMed
Summary

A new two-step calibration procedure accurately measures antibodies bound per cell (ABC) using Ultra Rainbow beads and biological standards. This method enhances multicolor flow cytometry analysis by providing a reliable scale for antibody quantification.

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Area of Science:

  • Immunology
  • Biotechnology
  • Analytical Chemistry

Background:

  • Accurate quantification of cell surface markers is essential in immunology and diagnostics.
  • Current multicolor flow cytometry calibration methods lack standardization for antibody binding quantification.

Purpose of the Study:

  • To present a robust two-step procedure for calibrating multicolor flow cytometers.
  • To establish a reliable method for quantifying antibody binding sites per cell (ABC).

Main Methods:

  • Calibrating fluorescence channels using Ultra Rainbow beads with assigned equivalent number of reference fluorophores (ERF) values.
  • Utilizing a biological standard with known antibody binding sites to translate ERF to ABC units.
  • Ensuring linear correlation between fluorescence signal and ERF values for bead populations.

Main Results:

  • Successfully established a two-step calibration procedure for multicolor flow cytometry.
  • Demonstrated the translation of the ERF scale to the antibodies bound per cell (ABC) scale using biological standards.
  • Validated the linearity between fluorescence signals and ERF values for bead populations.

Conclusions:

  • The presented procedure provides a scientifically sound foundation for accurate ABC quantification in flow cytometry.
  • This method offers improved accuracy and ease of implementation for multicolor flow cytometer calibration.
  • Future improvements in accuracy and user-friendliness of the calibration process are anticipated.