Labeling Adipose-Derived Stem Cells with Hoechst 33342: Usability and Effects on Differentiation Potential and DNA Damage

Figure 1

(a) Histogram of rat ASCs flow cytometry with antibodies against CD cell surface markers: the intensity of the fluorescence is shown on the -axis and the cell counts are shown on the -axis. The negative control (neg.) for PE- and FITC-conjugated antibodies (unstained cells) are shown in the upper right. An ASC-specific configuration with CD44, CD73, and CD90 being positive and CD31 and CD45 being negative was observed. (b) FDA-PI vitality staining: the percentage of vital cells is presented at day 1, 3, 7, 14, and 28 for control ASCs (“C”) and ASCs labeled with 0.5 μg/mL (“0.5”) and 5 μg/mL (“5”) H33342. Data is presented as the average of five experiments (error bars: SD). Normal vitality was observed for unlabeled cells and both types of labeled cells. Cells labeled with 5 μg/mL Hoechst showed slightly decreased vitality after 7 and 14 days. (c) MTT assay: the viability index is shown for both labeled groups until day 28. Data is presented as the average of five experiments (error bars: SD). (d) Phase contrast microscopy of rat ASCs used in this study. Scale bar represents 200 μm. (e) Cell counts at day 3, 7, 14, 21, and 28 for labeled (0.5 μg/mL and 5 μg/mL H33342) and unlabeled ASCs. Data is presented as the average of five experiments (error bar: SD). (f and g) Comet assay: (f) the olive tail moment (OTM) as a measure of DNA damage is shown 24 hours and 4 weeks after labeling with 0.5 μg/mL (“0.5”) H33342 and 5 μg/mL (“5”) and for unlabeled ASCs (“C”). Data is presented as the average of five experiments (error bar: SD); ns is not statistically significant, . Fluorescence images (g) of the comet assay of unlabeled (upper picture) and labeled ASCs (0.5 μg/mL lower left picture; 5 μg/mL lower right picture). The head of the comet (“H”, lower right picture) represents the undamaged DNA. A tail (“T”, lower right picture) is formed by damaged DNA and can be seen in particular after labeling with 5 μg/mL H33342, but not in unlabeled cells. Please notice that the lower right image depicts an over average tail value for illustration purposes.

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