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Stem Cells International
Volume 2016 (2016), Article ID 1652389, 14 pages
Research Article

Optimized Stem Cell Detection Using the DyeCycle-Triggered Side Population Phenotype

1Institute of Immunobiology, Kantonsspital St. Gallen, Rorschacherstrasse 95, 9007 St. Gallen, Switzerland
2Internal Medicine V, Medical University of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria
3Medical Clinic III for Oncology, Hematology and Rheumatology, University Clinic Bonn (UKB), Sigmund-Freud-Strasse 25, 53127 Bonn, Germany
4Tyrolean Cancer Research Institute, Innrain 66, 6020 Innsbruck, Austria

Received 14 July 2015; Accepted 31 August 2015

Academic Editor: Hannele T. Ruohola-Baker

Copyright © 2016 Maximilian Boesch et al. 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.

Supplementary Material

Supplemental Figure 1: Stability of DCV-Stained Samples. (A) SP-enriched A2780 cells were stained with 10 µM DCV (106 cells/ml) and kept on ice in the dark for 0, 6 and 22 h prior to flow cytometry. Even though cell death is increased after 6 and 22 h (data not shown), the staining itself (i.e., the separation of SP from NSP cells) is stable over the investigated time period. (B) Kinetic analysis of the same cells inhibited with 20 µM fumitremorgin C. Note that after washing, SP cells gradually re-appear owing to the henceforth lack of active inhibitor.

Supplemental Figure 2: SP/NSP Separation as a Function of Staining Duration. SP-enriched A2780 cells were stained with 10 µM DCV (106 cells/ml) for the indicated periods of time and analysed by flow cytometry. A staining duration of 30 min does not permit optimal dye accumulation in NSP cells, as detectable particularly in the long-wave range of DCV emission (‘DCV red’). Conversely, there is not much difference in staining outcomes between 60, 90 and 120 min, except that the NSP peak is somewhat sharpened with longer dye exposure. Altogether, and considering the heterogeneity in dye accumulation kinetics and cell death induction between different cell types, we propose a ‘default’ staining duration of 90 min, which can of course be adapted upon demand.

Supplemental Figure 3: Autofluorescence of Reserpine in the DCV-Relevant Wavelength Range. In the absence of DCV, A2780V cells were incubated for 90 min at 37°C with either no inhibitor, 50 µM verapamil, 20 µM fumitremorgin C, or 50 µM reserpine. Thereafter, cells were washed and analysed by flow cytometry for emission in the ‘DCV blue’ (450/50) and ‘DCV red’ (510/50) channels (note that due to the lack of DCV in the analysis, the detector voltages had to be increased accordingly). In contrast to verapamil and fumitremorgin C which are non-excitable by the violet laser, reserpine shows significant autofluorescence which might potentially interfere with the DCV signal, therefore making this compound less suited for DCV-based SP detection.

Supplemental Figure 4: Sox17 and EPC1 Expression in DCV-Defined SP/NSP Fractions. A2780V cells were stained with 10 µM DCV (2.5x106 cells/ml) and SP and NSP fractions were flow sorted (n=3). RNA was isolated and samples were processed for microarray analysis performed on the GeneChip® Human Gene 1.0 ST Array platform (Affymetrix). Data were normalized and bio-informatically analysed for differential gene expression as defined by M ≥±1 (representing a fold-change of ≥2) and p ≤0.05. Sox17 and EPC1, two stem cell-related genes downmodulated by Hoechst 33342, did not show underrepresentation in NSP cells, indicating that these genes are not regulated by DCV.

Supplemental Figure 5: Detection of DCV-SP Cells Using Different Filter Combinations. A2780V cells (106 cells/ml) were stained with 10 µM DCV and analysed on an LSRFortessa using the indicated filters. Evidently, all of the investigated combinations allow efficient discrimination of SP cells, indicating that DCV-based SP detection can be performed on various flow cytometric instruments without requiring change of filters.

  1. Supplementary Material