Effective Detection of Hoechst Side Population Cells by Flow Cytometry

Summary

Here, we show that high-power 375 nm and 405 nm lasers can effectively excite Hoechst 33342 and serve as a viable alternative to the 355 nm laser for side population (SP) cell detection, thereby expanding the range of available lasers in flow cytometry applications.

Abstract

The side population (SP) cells are identified through Hoechst 33342 staining and analyzed using flow cytometry (FCM). The Hoechst SP method is utilized for the isolation of stem cells based on the dye efflux properties of ATP-binding cassette (ABC) transporters. The method was initially employed for the identification and isolation of hematopoietic stem cells (HSCs), but it has now evolved to primarily focus on the identification and isolation of cancer stem cells (CSCs). The traditional detection method of FCM uses a 355 nm laser to excite the dye to detect SP cells. Through this study, we have successfully identified alternative approaches for dye excitation that can effectively replace the detection of SP cells using a 355 nm laser. This is achieved through the utilization of high-power 375 nm or 405 nm lasers. This allows us to exercise enhanced selectivity in the detection of SP cells rather than being solely limited to the 355 nm laser flow cytometry.

Introduction

The side population (SP) cells are identified through Hoechst 33342 staining and analyzed using flow cytometry (FCM). The SP cells are characterized by the pumping of the fluorescent DNA dye out of the cells through their ATP-binding cassette (ABC) transporter^1,2. The method was originally established for isolating murine bone marrow hematopoietic stem cells (HSCs)¹. The bone marrow SP cells were enriched with a population of HSCs characterized by  CD117⁺Sca-1⁺Lin^-Thy1^low expression^3,

Protocol

The experiments in this study utilized a total of 10 C57BL/6 mice aged between 8 and 12 weeks. Experimental operations were conducted in accordance with a protocol that was approved by the Institutional Animal Care and Use Committee of Sichuan University (#201609309). Experimental materials and the parameters of flow cytometry used in this article are listed in the Table of Materials.

1. Isolation and collection of mouse bone marrow cells

1. Euthanize mice in strict accordance with the institutional guidelines. To induce unconsciousness in the mouse, administer inhalation anesthetics starting with ....

Discussion

We used the protocol described to conduct three experiments, with each trial involving 3-4 mice, resulting in a total of 10 mice. The proportion of SP cells ranged from 0.05% to 0.76%. It is important to note that individual variations were observed among the mice. We utilized four flow cytometers to analyze the Hoechst samples. It is observed that the excitation of Hoechst dye by a 20 mW 355 nm laser on the new version of flow cytometry is equivalent to that of a 100 mW 355 nm laser on old-fashioned flow cytometry. This.......

Materials

Name	Company	Catalog Number	Comments
Automatic cell counter	Countstar	1M1200
Cell Filter(100 µm)	BIOFIL	CSS-013-100
Daily quality control fluorospheres	Beckman Coulter	B5230
Dulbecco's Modification of Eagle's Medium with 4.5g/L glucose (DMEM medium)	CORNING	10-013-CVRC
Fetal bovine serum	CORNING	35-081-CV
HBSS	Hyclone	SH30030.02
Hoechst 33342	Sigma-Aldrich	B2261
Propidium iodide (PI)	Sigma-Aldrich	P4170
Red blood cell lysis buffer	Beyotime	C3702
Verapamil	Sigma-Aldrich	V4629