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Related Experiment Video

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Protocol for culturing and functionally manipulating planarian neoblasts using SiR-DNA-based flow cytometry.

Wenya Zhang1, Xinran Li2, Yun Zhao3

  • 1Westlake Laboratory of Life Sciences and Biomedicine, Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310024, China; Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, Zhejiang 310024, China; School of Basic Medical Sciences, Fudan University, Shanghai 200030, China.

STAR Protocols
|February 7, 2024
PubMed
Summary

This study introduces a new method for culturing and manipulating planarian neoblasts, the stem cells responsible for regeneration. The novel SiR-DNA flow cytometry protocol avoids cell cycle inhibition, enabling better research into neoblast pluripotency and regeneration.

Keywords:
Cell cultureCell isolationDevelopmental biologyModel OrganismsStem Cells

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Area of Science:

  • * Developmental Biology
  • * Stem Cell Biology
  • * Molecular Biology

Background:

  • * Neoblasts are the sole proliferating cells in planarians, crucial for regeneration.
  • * Traditional Hoechst-based flow cytometry inhibits the cell cycle, limiting neoblast study.
  • * A non-inhibitory method is needed to investigate neoblast function and pluripotency.

Purpose of the Study:

  • * To present a novel protocol for culturing and functionally manipulating planarian neoblasts.
  • * To utilize SiR-DNA-based flow cytometry for non-inhibitory neoblast analysis.
  • * To enable further research into planarian stem cell pluripotency and regeneration mechanisms.

Main Methods:

  • * Development of a SiR-DNA-based flow cytometry protocol for planarian neoblasts.
  • * Detailed steps for cell dissociation, staining, and flow cytometry.
  • * Procedures for neoblast collection, culture, and Nanoluciferase mRNA transfection.

Main Results:

  • * Successful implementation of SiR-DNA flow cytometry for planarian neoblasts without cell cycle inhibition.
  • * Establishment of a protocol for culturing and functional manipulation of neoblasts.
  • * Demonstrated feasibility of mRNA transfection for functional studies.

Conclusions:

  • * The SiR-DNA flow cytometry protocol provides a non-inhibitory method for studying planarian neoblasts.
  • * This protocol advances the ability to investigate neoblast pluripotency and regeneration.
  • * Facilitates future research into the molecular mechanisms underlying planarian regeneration.