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Cellular Oncology
Volume 28, Issue 1-2, Pages 37-53

Flow Cytometry of the Side Population: Tips & Tricks

Irene Sales-Pardo,1 Ariadna Avendaño,1 Vanessa Martinez-Muñoz,1 Marta García-Escarp,2 Raquel Celis,1 Phil Whittle,4 Jordi Barquinero,2 Joan Carles Domingo,3 Pedro Marin,1 and Jordi Petriz1

1Cryopreservation Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain
2Research Unit, Transfusion Center and Tissue Bank, Barcelona, Spain
3Biochemistry Department, University of Barcelona, Spain
4Instrument Support, DakoCytomation A/S, Glostrup, Denmark

Copyright © 2006 Hindawi Publishing Corporation and the authors. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Background: The Side Population (SP) has become an important hallmark for the definition of the stem cell compartment, especially in the detection of these cells and in their physical isolation by fluorescence-activated cell sorting (FACS). SP cells are CD34neg and were discovered using ultraviolet excitation based on the efflux of Hoechst 33342 (Ho342). Although the method works as originally described, we believe that this method is difficult for most investigators. First, because the ability to discriminate SP cells is based on the differential retention of Ho342 during a functional assay; second, because of the difficulties in setting the right experimental and acquisition conditions; and third, because the analysis of the acquired data requires an extensive expertise on flow cytometry to accurately detect the SP events. Methods: First of all and mainly for the SP application, the laser beam paths were exhaustively checked to ensure the lowest coefficients of variation. Blood suspensions were prepared by erythrocyte lysis with ammonium chloride and hematopoietic cells were labeled with Ho342. Results: The Ho342 concentration and the staining procedure are critical for the optimal resolution of the SP cells. Although UV laser alignment is very important to resolve the dim tail that outlines the SP, the problem with Ho342 excitation is not the Hoechst Blue emission, but rather the Hoechst Red's (because of the weak emission). Conclusions: Each laboratory must establish its own expected ranges based on its instrument and results may vary slightly due to instrument differences such as the narrowness of the band pass filters, laser power, laser emission wavelength, nozzle type, differential of pressure, light collection system (cuvette versus jet-in-air) and beam shaping optics.