I would like to measure ROS levels or other oxidative stress marker in culture media from human stem cells.
Does anyone have a protocol? Do I have to use fresh media or can I use also frozen media?
Many thanks
https://www.promega.com/resources/protocols/technical-manuals/101/ros-glo-h2o2-assay-protocol/ in Promega we trust!
I transcribe the working protocol I used, you should consider the adjustments to your type of culture.
Effect on reactive oxigen species (ROS) production:
DCFH-DA assay. Production of intracellular ROS from RAW
264.7 macrophage cells was evaluated by using the non-polar dye,
dichlorofluorescein diacetate (DCFH-DA). The dye diffuses into
cells, gets trapped by deacetylation, and in the presence of
hydrogen peroxide becomes oxidized to yield 29,79-dichlorofluorescein
(DCF). RAW 264.7 macrophages were plated as described
above. After 24 h at 37uC, medium was removed and DMEM
supplemented with 0.5% FBS, 100 U/mL penicillin, 100 mg/mL
streptomycin and 50 mg/mL polymyxin B was added. ApoA-I (0.1
or 1.0 mg/mL) was added after one hour of incubation. LPS was
used as a control. Six hours later, cells were washed with DMEM
and incubated in presence of 20 mM DCFH-DA for 30 min at
37uC. After an extra wash with warm PBS, 100 mL passive lysis
buffer (Promega, WI) were added to each well and fluorescence
was measured on a microplate reader with excitation and emission
filters centered at 485 and 525 nm, respectively.
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043755
Do you have access to electron paramagnetic resonance (EPR) spectroscopy instrument facility? If so, you can use spin probes such as CMH to measure the superoxide radical generation from the stem cells.
Hi Juliana,
Like Ajit said you maybe able to detect cellular ROS easier than media ROS. We also looked as ROS in this paper attached (http://www.nature.com/cddis/journal/v7/n3/full/cddis2015409a.html OR https://www.researchgate.net/publication/296698867_Dengue-induced_autophagy_virus_replication_and_protection_from_cell_death_require_ER_stress_PERK_pathway_activation).
I'm not sure what is the half life of ROS in media but maybe you can calibrate your method. Even if you freeze the media, if the ROS scavenging in the media is fast it would be pointless. I don't know if you can concentrate your media through lyophilization, give it a shot and see if you can see ROS better? I would be surprised if this works! if it does please let me know! Best wishes!
-ED
Article Dengue-induced autophagy, virus replication and protection f...
You can use Amplex Red with HRP +/- catalase in serum-free, colorless media such as RPMI w/o phenol red.
Thanks everybody for all your suggestions! I'll definitely look into all them!
think this is a good method to measure the production of superoxide radicals live
MitoSOX™ Red Mitochondrial Superoxide Indicator, for live-cell imaging
Código de catálogo: M36008
Molecular Probes™ Aplicaciones relacionadas: Cell Structure | Cell Viability, Proliferation & Function
M36008
10 x 50 µg
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