*2.3. Anti-Apoptotic E*ff*ect of Histochrome against H2O2-Induced Cell Death*

To investigate the anti-apoptotic effects of histochrome in hCPCs, cells were treated with 1 mM H2O2 for 4 h and cell apoptosis was evaluated using flow cytometry by staining with Annexin V and 7-aminoactinomycin D (7-AAD). Annexin V/7-AAD assay revealed that treatment with H2O2 significantly increased the percentage of apoptotic cells (+H2O2, -Histochrome; 14.3% ± 2.36%) compared to the percentage of apoptotic cells in the non-treated control group (-H2O2, -Histochrome; -8.7% ± 0.84%, -H2O2, +Histochrome; 5.6% ± 0.40%, Figure 3A). In contrast, pretreatment of hCPCs with 10 μM of histochrome significantly increased the percentage of viable cells (+H2O2, +Histochrome; 95.2% ± 0.40%, Figure 3A) while decreasing the number of apoptotic cells (+H2O2, +Histochrome; 2.4% ± 0.49%, Figure 3A). We also investigated the effect of histochrome on cell morphology using phase contrast microscopy (Figure 3B). H2O2 treatment caused abnormal morphology and reduced cell viability. However, pretreatment with histochrome attenuated the morphological change induced by H2O2. In addition, live cell imaging analysis suggested that pretreatment with histochrome under the H2O2-induced oxidative stress condition significantly increased the number of live cells in a dose-dependent manner (Figure 3C).

**Figure 3.** Anti-apoptotic effect of histochrome against H2O2-induced cell death. (**A**) hCPCs were pretreated with 10 μM of histochrome for 24 h and then exposed to 1 mM H2O2 for 4 h. Apoptotic cells were quantified by fluorescence-activated cell sorting (FACS) analysis with Annexin V / 7-AAD staining. \*\* *p* < 0.01; \*\*\* *p* < 0.001 versus -H2O2 -histochrome; ### *p* < 0.001 versus +H2O2 -histochrome. (**B**) Representative images showing the morphology of hCPCs pretreated with histochrome (0 μM, 5 μM, 10 μM, and 20 μM) in the presence of H2O2-induced oxidative stress. Morphology of hCPCs was observed by phase contrast microscope. Scale bar = 50 μm (**C**) Live cells were quantified by phalloidin (green fluorescence) intensity. \*\*\* *p* < 0.001 versus -H2O2 -histochrome; # *p* < 0.05 versus +H2O2 -histochrome. Scale bar = 100 μm, *n* = 3. Error bars indicate S.E.M.
