CSE and H2O2 were used to induce ROS production as previously reported [15 (link)]. CSE was prepared from two commercially available cigarettes containing 8.0 mg tar and 0.7 mg nicotine (Marlboro 20 class A; Philip Morris Korea, Inc., Seoul, Korea) as described previously [15 (link)]. Here, 2% CSE and 200 μM H2O2 were used to generate oxidative stress in cells without reducing cell viability. Cells (1 × 105 cells/well) were exposed to oxidative stressors, washed with PBS, and incubated with the oxidant-sensitive fluorescent probe 5-(and 6)-carboxy-2’,7’-dichloro-dihydrofluorescein diacetate (H2DCFDA; Invitrogen, Eugene, OR, USA) for 30 minutes. The cells were then trypsinised, washed, and resuspended in PBS. To measure fluorescence intensity, a flow cytometer (ELITE flow cytometer; Coulter Cytometry, Inc., Hialeah, FL, USA) was used, and fluorescent photos were taken using an IX71-F22PH inverted fluorescence microscope (Olympus Corp., Tokyo, Japan).
Ix71 f22ph inverted fluorescence microscope
Manufactured by Olympus
Sourced in Japan
The IX71-F22PH is an inverted fluorescence microscope manufactured by Olympus. It is designed for biological and medical research applications. The microscope features a 22 mm field of view and a phase contrast function for enhanced contrast in live cell imaging.
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2 protocols using ix71 f22ph inverted fluorescence microscope
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Quantifying Oxidative Stress in Cells
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Quantifying Cellular Oxidative Stress
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Levels of ROS were measured using 5-(and 6)-carboxy-2 0 ,7 0dichlorodihydrofluorescein diacetate (H 2 DCFDA), an oxidantsensitive fluorescent probe. 17 Cells were seeded at a density of 5 3 10 5 cells per well in six-well plates to a total final volume of 2 mL and then treated with 2% CSE or 10 lM H 2 O 2 for 30 minutes. To evaluate the effect of resveratrol on ROS levels, cells were pretreated with 30 lM or 50 lM resveratrol for 24 hours. After the medium was removed, cells were washed with PBS and incubated with 10 lM H 2 DCFDA at 378C for 30 minutes, and then 2% CSE or 10 lM H 2 O 2 was added for 30 minutes. Subsequently, cells were trypsinized, washed, and resuspended in PBS. Thereafter, fluorescence intensity was measured with an IX71-F22PH inverted fluorescence microscope (Olympus Corp., Tokyo, Japan) and flow-cytometric analysis was performed (ELITE flow cytometer; Coulter Cytometry, Inc., Hialeah, FL, USA). For each sample, more than 10,000 events were acquired. Cells were gated, and the analysis was performed using only live populations. Fluorescent cells were also examined microscopically (3100 magnification).
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