4.7.1. Cell Culture

RPMI-1640 medium supplemented with heat-inactivated FBS and antibiotics (penicillin and streptomycin) was used to culture the Vero cells. The cells were maintained in controlled environment (humidified, 5% CO2). Periodic subculture was continued and cells were subjected to experiments at its exponential growth phase.

#### 4.7.2. Cell Viability and Intracellular ROS Scavenging Activity in H2O2 Stimulated Vero Cells

Cells were seeded (1 × 10<sup>5</sup> cells/mL) and were incubated for 16 h, samples were treated and incubated for 1 h. Following the cells were stimulated with H2O2 (1 mM). The cell viability was measured given 24 h incubation time using the MTT assay [51]. The intracellular ROS scavenging potential of the samples was measured using the dichloro- fluorescein diacetate (DCF-DA) assay [52]. Initially, the cells were seeded (1 × 10<sup>5</sup> cells/mL), incubated and treated with different sample concentrations. Cells were stimulated with H2O2 given 1 h incubation time and DCF-DA (500 μg/mL, stock) was treated to each well. The results were detected as a fluorescence measurement (Ex-485 nm, Em-530 nm) with a microplate reader (BioTech, Winooski, VT, USA).

#### 4.7.3. H2O2 Induced Cell Apoptosis through Nuclear Staining

The cells were seeded as explained above, treated with samples and was induced with H2O2. Following a 24 h incubation period, the cells were stained with cell permeable DNA dye Hoechst 33342 (10 μg/mL). Given 10 min incubation period, the cells were observed by a fluorescence microscope equipped with a CoolSNAP-Pro color digital camera (Olympus, Tokyo, Japan) [53,54].
