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. Author manuscript; available in PMC: 2022 Aug 7.
Published in final edited form as: Cell Rep. 2020 Jun 9;31(10):107737. doi: 10.1016/j.celrep.2020.107737

Figure 4. Low Phototoxicity during Continuous Optogenetic Stimulation of hESC Cultures.

Figure 4.

(A) Temperature of media after 24 h of continuous illumination at indicated light intensities. Each point represents the measurement from a single well.

(B) Bright-field images (top) of live wild-type hESC cultures illuminated at indicated light intensities after 48 h. Flow cytometry results (bottom) for Annexin V and propidium iodide (PI) stain of hESCs following 48 h of illumination. Positive gating indicated by vertical line. Scale bar, 250 μm.

(C) Quantification of Annexin V and PI shows a significant increase in apoptosis and cell death >1 μW/mm2 illumination intensity (pA = 0.0008, pPI = 0.39 at 0 versus 1 μW/mm2; pA = 0.0002, pPI = 0.009 at 0 versus 2 μW/mm2). No difference was observed between 0 and 0.5 μW/mm2 (pA = 0.61, pPI = 0.18). ANOVA followed by Tukey test. The graph shows means ± 1 SDs, n = 3 biological replicates.

(D) Endpoint cell count of wild-type and optoWnt hESCs after 48 h of continuous illumination at 0 or 0.8 μW/mm2. The graph shows means ± 1 SDs, n = 3 replicates. ANOVA followed by Tukey test.