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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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Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction
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Flow cytometry analysis using the Becton Dickinson FACS Calibur.

Kevin L Holmes1, Gillis Otten, Wayne M Yokoyama

  • 1National Institute of Allergy and Infectious Disease, Bethesda, Maryland, USA.

Current Protocols in Immunology
|April 25, 2008
PubMed
Summary

This guide explains how to operate the FACS Calibur flow cytometer for cell analysis, focusing on data acquisition. It covers single-color, two-color, three-color, and four-color immunofluorescence techniques for effective cell analysis.

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Area of Science:

  • Cell Biology
  • Immunology
  • Biotechnology

Background:

  • Flow cytometry is a crucial technique for cell analysis.
  • The FACS Calibur is a widely used instrument for flow cytometry.
  • Efficient operation is key to reliable experimental results.

Purpose of the Study:

  • To provide a comprehensive guide on operating the FACS Calibur flow cytometer.
  • To detail data acquisition procedures for various immunofluorescence applications.
  • To serve as a resource for users of BD flow cytometry instruments.

Main Methods:

  • Detailed operation procedures for the FACS Calibur.
  • Step-by-step protocols for single, two, three, and four-color immunofluorescence.
  • Methods for instrument performance checks and sensitivity assessment.
  • Live/dead cell discrimination using propidium iodide (PI).

Main Results:

  • Successful data acquisition using fluorescein isothiocyanate (FITC), phycoerythrin (PE), and allophycocyanin (APC) conjugated antibodies.
  • Demonstration of simultaneous multi-color analysis.
  • Validation of instrument performance and sensitivity.

Conclusions:

  • The FACS Calibur, with CELLQuest software, enables robust multi-color flow cytometric analysis.
  • This guide enhances user proficiency in data acquisition for diverse cell analysis applications.
  • The principles discussed are applicable to various BD flow cytometers.