Protocol - Reactive oxigen species (ROS) assay on HaCaT cells
UVB-induced ROS intracellular levels after Resveratrol pretreatment
Category:
Cell Biology
Last revision: Jan 29, 2015
Author(s):
Kisslinger A, Tramontano D.
Contact
Name: Kisslinger Annamaria
Address: IEOS -CNR via Pansini 5, 80131 Naples Italy
Phone: +3908107464552
Email: a.kisslinger@ieos.cnr.it
Steps
| Description | Temperature | Time | Note |
|---|---|---|---|
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80% confluent cells
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37°C,5% CO2 in a humidified incubator
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seed 24hours before treatment in 24-well plates
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|
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Add Resveratrol
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37°C,5% CO2 in a humidified incubator
|
from 2 to 24 hours
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25 and 100 µM
|
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Remove medium after incubation
|
RT
|
||
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Wash twice with PBS Buffer
|
RT
|
||
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Irradiate cells with UVB source
|
RT
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10 to 100 mJ/cm2
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without plastic lid
|
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Load the cells with H2DCF-DA
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37°C,5% CO2 in a humidified incubator
|
15 min
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10 µM in PBS Buffer
|
|
Washed twice PBS buffer
|
RT
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in the dark
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|
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Read plates in a fluorescent microplate reader
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RT
|
excitation wavelength of 485 nm and an emission wavelength of 538 nm
|
Other informations
Irradiating source consists of three lamps (Philips Ultraviolet 8 TL 20W/01 RS lamps; Philips, Eindhoven, Netherlands) generating UVB light in the range of 290–320 nm with an emission peak at 312 nm. Intensity of UVB irradiation was measured using a phototherapy radiometer (International Light, Newburyport, MA).
Microplates Reader: Perkin Elmer LS 55 Luminescence Spectrometer, Perkin-Elmer Ltd., Beaconsfield, England). Fluorescence was monitored using an excitation wavelength of 485 nm and an emission wavelength of 538 nm
Quality validation:
Yes
Validation info
The protocol has been published in a peer-reviewed journal.
