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.
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