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Flow cytometry of the Side Population: tips & tricks.
Irene Sales-Pardo1, Ariadna Avendaño, Vanessa Martinez-Muñoz
1Cryopreservation Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain.
Summary
Identifying side population (SP) stem cells using Hoechst 33342 (Ho342) dye via fluorescence-activated cell sorting (FACS) can be challenging. Optimizing Ho342 concentration and staining is crucial for accurate SP cell detection and isolation.
Area of Science:
- Biotechnology
- Cell Biology
- Hematology
Background:
- Side Population (SP) cells are a key indicator of stem cell populations.
- SP cells are identified by their ability to efflux Hoechst 33342 (Ho342) dye.
- Current methods for SP cell detection using fluorescence-activated cell sorting (FACS) can be complex and require specialized expertise.
Purpose of the Study:
- To address the challenges in identifying and isolating SP cells using Ho342 dye and FACS.
- To provide insights into optimizing the SP cell detection protocol.
Main Methods:
- Ensured optimal laser beam paths for low coefficients of variation.
- Prepared blood cell suspensions using erythrocyte lysis.
- Labeled hematopoietic cells with Ho342 dye.
Main Results:
- Ho342 concentration and staining procedures are critical for resolving SP cells.
- UV laser alignment is important, but weak Hoechst Red emission poses a challenge.
- Optimizing these parameters is essential for accurate SP cell identification.
Conclusions:
- Laboratory-specific optimization is necessary for consistent SP cell detection.
- Instrument variations can influence results, requiring individual range establishment.
- Standardization of protocols may improve reproducibility across different labs.