Realtime glotm mt cell viability assay

The treated samples, including control (no treatment), RESV only, EP only, and EP + RESV, at 1.0 × 10⁶ cells/mL concentration were seeded into 96-well plates, per Promega’s protocol. The cells were supplied with 55 μL of prepared media in each well. They are then incubated with optimal growth conditions for 24–60 h. To study the cell viability, Real-Time-Glo^TM MT Cell Viability Assay (G9711, Promega, USA) was used. Synergy LX Multi-Mode Reader (SLXATS, BioTek Instruments, USA) was utilized for recording the luminescence (Lum). Equation 1 was used to calculate the percentage of cell viability. The experimental workflow is shown in Figure 2.
Equation 1:
$$Cell\hspace{0.17em}viability\hspace{0.17em}(\%)=\frac{Treated\hspace{0.17em}cell\hspace{0.17em}Lum\hspace{0.17em}value}{Control\hspace{0.17em}Lum}\times 100\%$$

Cell cytotoxicity was determined by measuring ATP content using the RealTime-Glo^TM MT Cell Viability Assay (Promega, Milan, Italy) according to the manufacturer’s instructions. Briefly, 500 cells were plated in 96-well plates in 200 μL of cell medium (RPMI for SKOV-3 and DMEM/DMEM F12 1:1 for hASC). After 24 h, the cells were exposed to increasing concentrations of nanoconjugated or non-conjugated teicoplanin or to the corresponding concentrations of NPs (considering the teicoplanin loaded per mg of NPs) and then a solution 2× the substrate and NanoLuc^® Enzyme were added. The cells were incubated at 37°C and in 5% CO₂-humidified atmosphere, and luminescence was read every 24 h using the Infinite F200 plate reader (Tecan Group, Männedorf, Switzerland).

The cytotoxicity of RES, A-1, and A-3 was measured using the RealTime-Glo^TM MT Cell Viability Assay (Promega, Madison, WI, USA). The cells (10,000 cells/well) were seeded into 96-well plates. After 24 h, they were treated with various 10-fold dilutions of RES, A-1, and A-3 with concentrations ranging from 0.1 µM to 1000 µM. Cells with 0.1% DMSO (ATCC; Manassas, VA, USA) were included as a control. Luminescence was measured every 12 h for 72 total hours in a Cytation^TM 5 (BioTek, Winooski, VT, USA) plate reader. All experiments were performed in triplicates. The IC₅₀ (concentration of compound exhibiting 50% of cell growth inhibition) was attained from standard curves of the cell viability percentage vs. log concentration of compounds using GraphPad Prism 9(San Diego, CA, USA). Based on the IC₅₀ of each compound and lower toxicity to MCF-10A, the most effective compound was subjected to further examination.