Flow cytometry of the Side Population: Tips & tricks
Article type: Research Article
Authors: Sales-Pardo, Irene; | Avendaño, Ariadna; | Martinez-Muñoz, Vanessa | García-Escarp, Marta | Celis, Raquel | Whittle, Phil | Barquinero, Jordi | Domingo, Joan Carles | Marin, Pedro | Petriz, Jordi;
Affiliations: Cryopreservation Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain | Research Unit, Transfusion Center and Tissue Bank, Barcelona, Spain | Biochemistry Department, University of Barcelona, Spain | Instrument Support, DakoCytomation A/S, Glostrup, Denmark
Note: [] These two authors contributed equally to this work.
Note: [] These two authors contributed equally to this work.
Note: [] Corresponding author: Jordi Petriz, Institut de Recerca, Hospital Vall d'Hebron, Vall d'Hebron 119-129, E-08035 Barcelona, Spain. Tel.: +34 932 746 873; Fax: +34 934 894 015; E-mail: [email protected].
Abstract: 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.
Keywords: Side Population, stem cells, CD34-negative, Hoechst 33342, flow cytometry
Journal: Analytical Cellular Pathology, vol. 28, no. 1-2, pp. 37-53, 2006
