Flow Cytometry of the Side Population: Tips & Tricks
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...
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| Format: | Article |
| Language: | English |
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Wiley
2006-01-01
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| Series: | Cellular Oncology |
| Online Access: | http://dx.doi.org/10.1155/2006/536519 |
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| author | Irene Sales-Pardo Ariadna Avendaño Vanessa Martinez-Muñoz Marta García-Escarp Raquel Celis Phil Whittle Jordi Barquinero Joan Carles Domingo Pedro Marin Jordi Petriz |
| author_facet | Irene Sales-Pardo Ariadna Avendaño Vanessa Martinez-Muñoz Marta García-Escarp Raquel Celis Phil Whittle Jordi Barquinero Joan Carles Domingo Pedro Marin Jordi Petriz |
| author_sort | Irene Sales-Pardo |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-0bc7a8dfed044c52882239901f5c1dee |
| institution | Kabale University |
| issn | 1570-5870 1875-8606 |
| language | English |
| publishDate | 2006-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Cellular Oncology |
| spelling | doaj-art-0bc7a8dfed044c52882239901f5c1dee2025-02-03T01:12:29ZengWileyCellular Oncology1570-58701875-86062006-01-01281-2375310.1155/2006/536519Flow Cytometry of the Side Population: Tips & TricksIrene Sales-Pardo0Ariadna Avendaño1Vanessa Martinez-Muñoz2Marta García-Escarp3Raquel Celis4Phil Whittle5Jordi Barquinero6Joan Carles Domingo7Pedro Marin8Jordi Petriz9Cryopreservation Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, SpainCryopreservation Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, SpainCryopreservation Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, SpainResearch Unit, Transfusion Center and Tissue Bank, Barcelona, SpainCryopreservation Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, SpainInstrument Support, DakoCytomation A/S, Glostrup, DenmarkResearch Unit, Transfusion Center and Tissue Bank, Barcelona, SpainBiochemistry Department, University of Barcelona, SpainCryopreservation Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, SpainCryopreservation Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, SpainBackground: 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.http://dx.doi.org/10.1155/2006/536519 |
| spellingShingle | Irene Sales-Pardo Ariadna Avendaño Vanessa Martinez-Muñoz Marta García-Escarp Raquel Celis Phil Whittle Jordi Barquinero Joan Carles Domingo Pedro Marin Jordi Petriz Flow Cytometry of the Side Population: Tips & Tricks Cellular Oncology |
| title | Flow Cytometry of the Side Population: Tips & Tricks |
| title_full | Flow Cytometry of the Side Population: Tips & Tricks |
| title_fullStr | Flow Cytometry of the Side Population: Tips & Tricks |
| title_full_unstemmed | Flow Cytometry of the Side Population: Tips & Tricks |
| title_short | Flow Cytometry of the Side Population: Tips & Tricks |
| title_sort | flow cytometry of the side population tips tricks |
| url | http://dx.doi.org/10.1155/2006/536519 |
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