**2.6. Cell morphological study**

**2.5. Microculture tetrazolium assay**

24 Current Understanding of Apoptosis - Programmed Cell Death

sue culture plate at a density of approximately 0.5 × 10<sup>4</sup>

% *of cell viability* = \_\_\_

were: 6.90, 5.10 and 3.77 μM Artonin E at 24, 48 and 72 h, respectively.

the plate by incubating at 37°C under 5% CO<sup>2</sup>

The colorimetric microculture tetrazolium assay ([7] was used to assess breast cancer cell viability. The tetrazolium dye is converted into an insoluble formazan by the action of nicotinamide adenine dinucleotide hydrogenase present in metabolically active cells. This cellular conversion into a purple-colored formazan is directly proportional to the number of viable cells. Briefly, exponentially growing cells were seeded into a 96-well flat bottom tis-

ment, the cells were incubated with the tested compounds at concentrations ranging from 1.56 to 100 μM. Control cells were treated with 0.01% of DMSO, which was equivalent to the amount of DMSO used as vehicle. After each of the 24, 48 and 72 h treatment time period, 20 μL of 5 mg/mL of MTT solution was added to each well and the plate was reincubated for 4 h to facilitate catalysis by mitochondrial dehydrogenases. Next, 100 μL DMSO was added to each well to solubilize the formazan crystals. The absorbances of the resultant solutions were determined colorimetrically at 570 nm. The experiment was performed in triplicate.

We conducted a nonlinear regression analysis and utilized the GraphPad Prism software to fit a dose-response curve. The concentration of the compound that triggered a 50% growth inhibition was indicated at different time interval in **Figure 1**. The percentage viability used in

**Figure 1.** Viability of MCF-7 cell line treated with Artonin E. The half maximum growth inhibitory concentrations (IC50)

*AT AC*

fitting the dose-response curve was calculated using the following formula:

cells/well and allowed to adhere to

× 100 (2)

and 95% air overnight. Following cell attach-

Acridine Orange (AO) and propidium iodide (PI) double staining assay was employed to determine the effect of Artonin E in the induction of breast cancer cell death. A total of 3 × 10<sup>5</sup> MCF-7 breast cancer cells were seeded in a six-well plate and exposed to various treatment concentrations of Artonin E (1.56–100 μM) at varying time points (24–72 h). The adherent MCF-7 cells were trypsinized, and centrifuged at 2000 rpm (Hettich Universal 32 R centrifuge, DJB Labcare Ltd., UK) for 5 min. The pellet was washed with ice-cold PBS, re-centrifuged before suspending in 20 μL of PBS. The cells were thereafter stained on ice with 20 μL dye containing 10 μg/mL of AO with 10 μg/mL of PI. Carl Zeiss Axioskop plus-2 fluorescence microscope was used to visualize aliquots of 20 μL of the cell suspension. Three fields each containing at least 200 cells were immediately assessed for viability, early and late apoptosis as well as necrosis [9]. The experiment was performed in triplicate.
