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Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos
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Cell Counting Techniques for Preimplantation Mouse Embryos Using Fluorescent DNA Dyes.

Virginia E Papaioannou1, Richard R Behringer2

  • 1Department of Genetics and Development, Columbia University Medical Center, New York, New York 10032, USA vep1@columbia.edu.

Cold Spring Harbor Protocols
|November 6, 2023
PubMed
Summary

Accurately counting cells in early embryos is challenging after morula compaction. This study presents protocols using fluorescent DNA dyes for precise nuclear counting in preimplantation embryos, enabling differentiation of trophectoderm and inner cell mass (ICM).

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Area of Science:

  • Developmental Biology
  • Embryology
  • Cell Biology

Background:

  • Accurate cell counting in preimplantation embryos is crucial for developmental studies.
  • Light microscopy becomes challenging for cell enumeration after morula compaction due to indistinct cell boundaries in living embryos.

Purpose of the Study:

  • To provide reliable protocols for quantifying cell numbers in preimplantation embryos.
  • To overcome limitations of morphological assessment in early embryonic development.

Main Methods:

  • Utilizing fluorescent DNA dyes (Hoechst, propidium iodide) for nuclear staining.
  • Implementing a protocol for simple nuclear counting using a single DNA dye.
  • Detailing a differential nuclear counting method for trophectoderm and inner cell mass (ICM) using immunosurgery and two DNA dyes.

Main Results:

  • Demonstrated the efficacy of fluorescent DNA dyes for accurate cell enumeration in preimplantation embryos.
  • Established a method for distinguishing and counting nuclei of the trophectoderm and ICM.

Conclusions:

  • Fluorescent DNA dye-based nuclear counting offers a robust alternative to morphological assessment for cell number determination in embryos.
  • The described protocols facilitate precise quantification of cell populations within the developing embryo.