I am culturing HUVEC up to 80% confluency (EGM-2; Lonza), then let them serum starved (EBM-2; Lonza) for 24hr and treated with TNF-a (10ng/ml) for 30min. With DCFDA I didn't get that much of increase in ROS. And the outcome of Lucigenin assay puzzeled me. Now I am not sure whether the TNF-a is really working in terms of ROS generation in HUVEC.
Am I doing the whole thing right?
You may need to check different doses of TNFalpha, may be upto 50ng/ml. Instead you may also try LPS with different doses
Hi Arif,
I would reccomend treating HUVECs for at least a couple of hours to generate ROS. 30 mins maybe be too less. Try more time points like 2 hrs, 4 hrs and 6 hrs.
Article The Role of Reactive Oxygen Species in TNFα-Dependent Expres...
Thank you Harsh and Deeksha.
I'll try 20ng/ml for 30min, 1hr, 6hr and 24hr.
Best,
Taufiq
I will suggest to use phenol red free medium with 10% serum to measure the ROS in TNF treated cells even after 20 minutes. 10 ng/ml TNF is more than enough to generate ROS.
There is no need to go for time duration, 24h. Even 6h is too long. Whatever, time and concentration combination you try; you need to check viability of your cells. 2h co-culture with a modest dose below 50ng may work.
My suggestion goes into another direction. I recommend not (never) to use DCFDA (see for ref. Winterbourn, C.). Instead use Amplex UltraRed assay and make single point measurements. If You decide to apply this method contact me under [email protected] for further information about the right protocol.
Monika
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