FIGURE 2.
HS680 fluorescent imaging agent labels hypoxic MSC in large aggregates. A, Merged confocal images of DIC and fluorescence of U87‐MG cells and MSC incubated with HS680 for 24 h in 2D culture. B, spectral unmixing analysis of 3D multicellular aggregates of U87‐MG cells and MSC cultivated for 3 d in hanging drops in the presence of HS680 dye. This analysis was performed using the IVIS Spectrum system and Living Image 4.5 software, and allowed to separate the autofluorescence signal from the specific signal of HS680 dye. C, The correlation between aggregate diameter and hypoxia level in MSC spheroids formed by hanging‐drop culture of increasing numbers of MSC. The values are mean ± SEM of at least 10 spheroids per experimental condition. D, quantification of hypoxia of in vivo MSC aggregates at 7 d after subcutaneous injection of 1 × 106 or 3.5 × 106 cells (measured as HS680‐specific signal). Data are shown as mean ± SEM of n = 5 mice. (**P < .01, Student's t test)