Fig. 3

Organoid forming efficiency, viability, and fluorescence signal durability. a Fluorescence signal intensity immediately after staining with CMFDA (5–25 μM), determined by flow cytometry. b Decline in fluorescence signal intensity (15 μM CMFDA) over time. c, d PrestoBlue^TM viability assay performed consecutively c 24 h and d 48 h after staining with CMFDA (5–25 μM). Arbitrary unit (AU). No statistically significant (ns) difference in viability was detected. e Organoid forming efficiency determined 10 days following single-cell seeding of CMFDA-stained (5–25 μM) and unstained cells. All values were normalized to the mean of the unstained DMSO-control. A statistically significant increase was observed with 5 μM (*) of CMFDA (p = 0.03) (CMFDA median = 1.034% ICR = 0.998–1.186%, DMSO median = 1.003% ICR = 0.976–1.024%). A drastic and statistically significant reduction of the organoid forming efficiency was observed with increasing concentrations of with CMFDA 15 μM (**) (median = 0.75% ICR = 0.710–0.797%) and 25 μM (***) (median = 0.017% ICR = 0.013–0.024%). f Brightfield and fluorescent images of organoids at day 0 and day 3 after staining with CMFDA (15 μM), along with unstained controls, demonstrating maintained growth capacity. White scale bar, 100 μm