ZER-induced cell death in HeLa cancer cells
was quantified using propidium iodide (PI) and acridine-orange (AO) double
staining according to standard procedures and examine under fluorescence microscope
(Lieca attached with Q-Floro Software) [18 , 19 (link)]. Briefly, treatment was carried
out in a 25 mL culture flask (Nunc). HeLa cells were plated at concentration of
1 × 106 cell/mL, and treated with ZER at IC50 concentration. Flasks were incubated in atmosphere of 5% CO2 at 37°C for 24, 48, and 72 hours. The cells were then spun down at 1000 rpm for 10 minutes.
Supernatant was discarded and the cells were washed twice using phosphate
buffer saline (PBS) after centrifuging at 1000 rpm for 10 minutes to remove the
remaining media. Ten microliters of fluorescent dyes containing acridine orange
(AO, 10 μg/mL) and propidium iodide (PI, 10 μg/mL) were added into the cellular pellet at equal
volumes of each. Freshly stained cell suspension was dropped into a glass slide
and covered by coverslip. Slides were observed under UV-fluorescence microscope
within 30 minutes before the fluorescence color starts to fade. The percentages
of viable, apoptotic, and necrotic cells were determined in more than 200
cells. Acridine orange (AO) and propidium iodide (PI) are intercalating nucleic
acid specific fluorochromes which emit green and orange fluorescences,
respectively, when they are bound to DNA. Of the two, only AO can cross the
plasma membrane of viable and early apoptotic cells. Viewed by fluorescence
microscopy, viable cells appear to have green nucleus with intact structure
while apoptotic cells exhibit a bright-green nucleus showing condensation of
chromatin as dense green areas. Late apoptotic cells and necrotic cells will
stain with both AO and PI. Comparatively, PI produces the highest intensity
emission. Hence, late apoptotic cells exhibited an orange nucleus showing
condensation of chromatin whilst necrotic cells display an orange nucleus with
intact structure. This assay provides a useful quantitative evaluation and was
done three times (n = 3).
was quantified using propidium iodide (PI) and acridine-orange (AO) double
staining according to standard procedures and examine under fluorescence microscope
(Lieca attached with Q-Floro Software) [18 , 19 (link)]. Briefly, treatment was carried
out in a 25 mL culture flask (Nunc). HeLa cells were plated at concentration of
1 × 106 cell/mL, and treated with ZER at IC50 concentration. Flasks were incubated in atmosphere of 5% CO2 at 37°C for 24, 48, and 72 hours. The cells were then spun down at 1000 rpm for 10 minutes.
Supernatant was discarded and the cells were washed twice using phosphate
buffer saline (PBS) after centrifuging at 1000 rpm for 10 minutes to remove the
remaining media. Ten microliters of fluorescent dyes containing acridine orange
(AO, 10 μg/mL) and propidium iodide (PI, 10 μg/mL) were added into the cellular pellet at equal
volumes of each. Freshly stained cell suspension was dropped into a glass slide
and covered by coverslip. Slides were observed under UV-fluorescence microscope
within 30 minutes before the fluorescence color starts to fade. The percentages
of viable, apoptotic, and necrotic cells were determined in more than 200
cells. Acridine orange (AO) and propidium iodide (PI) are intercalating nucleic
acid specific fluorochromes which emit green and orange fluorescences,
respectively, when they are bound to DNA. Of the two, only AO can cross the
plasma membrane of viable and early apoptotic cells. Viewed by fluorescence
microscopy, viable cells appear to have green nucleus with intact structure
while apoptotic cells exhibit a bright-green nucleus showing condensation of
chromatin as dense green areas. Late apoptotic cells and necrotic cells will
stain with both AO and PI. Comparatively, PI produces the highest intensity
emission. Hence, late apoptotic cells exhibited an orange nucleus showing
condensation of chromatin whilst necrotic cells display an orange nucleus with
intact structure. This assay provides a useful quantitative evaluation and was
done three times (n = 3).
