OxiSelect™ Intracellular ROS Assay Kit (Green Fluorescence)

Protocol tips

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- 1X104 cells were seeded and incubate overnight at 37C.	- Cells were treated with DCFH-DA in 100 μL/well for 45 min at 37°C.

Upstream tips
- 1X104 cells were seeded and incubate overnight at 37C.
Protocol tips
- Cells were treated with DCFH-DA in 100 μL/well for 45 min at 37°C.

Publication protocol

Intracellular ROS generation was assessed using the ROS assay kit. A total of 1X104 cells were
seeded in a 96-well plate and incubated at 37°C overnight. Following overnight incubation, cells
were washed with PBS 3 times and incubated with 2’,7’-dichlorodihydrofluorescin diacetate
(DCFH-DA), which is a fluorescent cell compound, at volumes of 1X DCFH-DA in 100 μL/well
for 45 min at 37°C. The DCFH-DA solution was removed, and the cells were washed 3 times
with PBS. Dose-response analyses were conducted on pancreatic cancer cell lines (HPAC, MIA
PaCa-2 and PANC-1) with various concentrations of nimbolide (1, 3, 5, 10, 15, 20 and 25 µM)
for 20 min. Similarly, a time-course analysis was performed for 20, 60, 120, 180 and 360
minutes with IC50 concentrations of nimbolide respective to each cell line. After the treatments, 3
washes with PBS were performed, and 1x cell lysis buffer was added and mixed thoroughly for 5
min. Then, 150 µL of the mix was transferred to a black cell culture fluorometric 96-well plate
for fluorescence measurement. Using a BMG LabTech fluorescence plate reader, the results were
read at 480 nm excitation and 530 nm emission. Fluorescence of the samples was also analyzed
and captured using a Nikon laser scanning confocal microscope (Nikon Instruments, Melville,
NY, USA)

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Manufacturer protocol

Download the product protocol from Cell Biolabs for OxiSelect™ Intracellular ROS Assay Kit (Green Fluorescence) below.

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