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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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Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization
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Multi-parametric flow cytometric cell cycle analysis using TO-PRO-3 iodide (TP3): detailed protocols.

Michele Tavecchio1, Matteo Simone, Sergio Bernasconi

  • 1Flow Cytometry Unit, Department of Oncology, Mario Negri Institute for Pharmacological Research, Milan, Italy.

Acta Histochemica
|December 28, 2007
PubMed
Summary

TO-PRO-3 iodide (TP3) offers an alternative to propidium iodide (PI) for DNA analysis in flow cytometry. TP3 provides excellent DNA histograms and simplifies multi-parametric analysis by eliminating the need for fluorescence compensation.

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Area of Science:

  • Flow cytometry
  • Cell cycle analysis
  • Nucleic acid staining

Background:

  • Propidium iodide (PI) is the standard DNA stain for flow cytometry.
  • Optimizing protocols for DNA analysis is crucial for accurate cell cycle assessment.
  • Cyanine dyes offer potential alternatives with distinct spectral properties.

Purpose of the Study:

  • To establish an optimal protocol for TO-PRO-3 iodide (TP3) in flow cytometric DNA analysis.
  • To evaluate TP3 as an alternative to PI for cell cycle studies.
  • To assess the utility of TP3 in multi-parametric flow cytometry.

Main Methods:

  • Testing TP3 in monocytes and various cell lines.
  • Investigating effects of fixatives, dye concentrations, incubation times, and RNAse.
  • Evaluating DNA histograms for G1 peak CV, G2/G1 ratio, and debris.
  • Assessing TP3 in combination with other fluorochromes like FITC and PE.

Main Results:

  • Optimal TP3 concentrations ranged from 0.1 to 2 microM, showing a linear increase in G1 peak position.
  • 70% ethanol or 1% methanol-free formaldehyde followed by 70% ethanol yielded the best DNA histograms.
  • Incubation with 0.5 microM TP3 plus RNAse for 30 min provided excellent results across most cell lines.
  • TP3 signals do not require fluorescence compensation when used with other fluorochromes.

Conclusions:

  • TO-PRO-3 iodide (TP3) is a viable alternative to propidium iodide (PI) for DNA analysis.
  • Optimized TP3 protocols yield high-quality DNA histograms suitable for cell cycle analysis.
  • TP3 simplifies multi-parametric flow cytometry by eliminating the need for fluorescence compensation.