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Updated: Jul 6, 2026

Effective Detection of Hoechst Side Population Cells by Flow Cytometry

Effective Detection of Hoechst Side Population Cells by Flow Cytometry

Published on: August 23, 2024

Isolation and functional characterization of side population stem cells.

Jonathan B Johnnidis¹, Fernando D Camargo

¹Whitehead Institute for Biomedical Research, Cambridge, MA, USA.

Methods in Molecular Biology (Clifton, N.J.)

|March 29, 2008

Summary

Primitive cells possess a unique "side population" (SP) phenotype, enabling them to efflux Hoechst 33342 dye. This characteristic allows for the isolation of hematopoietic stem cells (HSCs) from bone marrow (BM) using flow cytometry.

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Area of Science:

Stem cell biology
Hematopoiesis
Flow cytometry

Background:

The side population (SP) phenotype is characterized by the ability of primitive cells to efficiently efflux Hoechst 33342 dye.
In bone marrow (BM), the SP fraction is enriched for hematopoietic stem cells (HSCs).
Isolating functional HSCs is crucial for understanding stem cell biology and developing regenerative therapies.

Purpose of the Study:

To describe a reproducible protocol for isolating murine bone marrow (BM) side population (SP) cells.
To present analytic methods for assessing the functionality of SP-derived stem cells.
To provide a basis for the isolation and functional assessment of primitive hematopoietic stem cells.

Main Methods:

Utilizing flow cytometry to identify and isolate side population (SP) cells based on Hoechst 33342 dye efflux.

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Employing single cell transplantation assays to evaluate the stem cell potential and functionality of isolated SP cells.

Detailed description of the protocol for reproducible murine bone marrow (BM) SP cell isolation.

Main Results:

A reproducible method for isolating murine bone marrow (BM) side population (SP) cells was established.
The SP fraction was confirmed to be highly enriched for hematopoietic stem cells (HSCs).
Functional assessment using single cell transplantation validated the stem cell capacity of SP-derived cells.

Conclusions:

The side population (SP) phenotype serves as a reliable marker for isolating primitive hematopoietic stem cells (HSCs) from murine bone marrow (BM).
The described protocol enables reproducible isolation of SP cells for further functional studies.
Assessment of SP-derived stem cell functionality is achievable through methods like single cell transplantation.