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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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Related Experiment Video

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Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors
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Flow sorting from organ material by intracellular markers.

Ulrik Moerch1, Henriette S Nielsen, Dorthe Lundsgaard

  • 1Department of Virology and Molecular Toxicology, Novo Nordisk A/S, Maaloev, Denmark.

Cytometry. Part a : the Journal of the International Society for Analytical Cytology
|June 2, 2007
PubMed
Summary

This study presents a new Fluorescence-Activated Cell Sorting (FACS) protocol for isolating rare cells using intracellular markers. The method is compatible with downstream analysis of proteins, peptides, and mRNA.

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Area of Science:

  • Endocrinology
  • Cell Biology
  • Molecular Biology

Background:

  • Fluorescence-activated cell sorting (FACS) is valuable for studying rare cell populations.
  • Intracellular markers offer an alternative when surface markers are unknown.
  • Current methods face challenges due to potential protein modification and RNA degradation from fixation/permeabilization.

Purpose of the Study:

  • To develop a robust FACS protocol for sorting rare cells based on intracellular markers.
  • To enable downstream molecular analysis of sorted rare cells.

Main Methods:

  • Utilized rat thyroid gland tissue for model system.
  • Employed immunostaining for intracellular calcitonin peptide and thyroglobulin.
  • Sorted rare (parafollicular) and frequent (follicular) endocrine cell types.

Main Results:

  • Successfully sorted rare and frequent endocrine cell types based on intracellular protein expression.
  • Sorted cells were suitable for Western blot analysis.
  • Sorted cells were compatible with immunoassay detection of calcitonin peptide hormone.
  • Sorted cells yielded viable RNA for RT-PCR analysis.

Conclusions:

  • Developed a robust FACS protocol for intracellular marker-based cell sorting.
  • The protocol supports downstream analysis of proteins, peptides, and mRNA.
  • This method enhances the study of rare cell populations from dissociated organ material.