Are you sure that TNF should work in these cells? On the other hand , have you a positive control for ROS o nitric oxide assays? The second question is because cells may be OK but the problem may be in the assays. Those assays are not "easy"

Hi, thanks for answering!

Yes, TNF-alpha stimulates these cells. We can see that at these articles:

1 - Hericium erinaceus Inhibits TNF-𝛼-Induced Angiogenesis and

ROS Generation through Suppression of MMP-9/NF-𝜅B

Signaling and Activation of Nrf2-Mediated Antioxidant Genes in

Human EA.hy926 Endothelial Cells

2 - TNFa induces the expression of genes associated with endothelial dysfunction through p38MAPK-mediated down-regulation of miR-149

3 - TNF affects energy metabolism and stimulates biogenesis of mitochondria in EA.hy926 endothelial cells

4- PPARa and PPARd activators inhibit cytokine-induced nuclear translocation of NF-kB and expression of VCAM-1 in EAhy926 endothelial cells

5- Vaspin inhibited proinflammatory cytokine induced activation of nuclear factor-kappa B and its downstream molecules in human endothelial EA.hy926 cells

6 - The Role of Haem Oxygenase-1 in the Decrease of Endothelial Intercellular Adhesion Molecule-1 Expression by Curcumin

TNF-alpha is supposed to stimulate the production of ROS, Nitric Oxide, Adhesion Molecules and other pro inflammatory components, such as cytokines and chemokines. These assays have been standardized in our laboratory a long time ago, and they resulted in several publications. But I'll try some adjustments and see if it works.

Again, thank you so much for your answer!!

Regards, 

Jordana.

Dear Jordana,

did you arrest the cells with SFM before the stimulation? Usually, cells need to be growth synchronized by serum deprivation (24-48 h) to get expected response. Another technical issue is if you perform the experiment with or without serum? I suggest to check ROS and NO production in serum-free conditions. 

Good luck

Hi, Krzysztof! Thanks for answering!

Well, I seed them at the plate with 2% serum and then I remove this media and treat them with TNF-alpha without serum. Do you think that would be better If I seed them without the serum too? Before my experiments I seed the cells for 17-18 hours before treatment.

Regards, Jordana.

I think you should allow the cells to adapt to culture conditions and maintain them for a while in 10% serum. I suggest to keep them until 80% confluency. Afterwards remove serum and synchronize them for 24 h before exposure to TNF or LPS. Then use the stimuli in serum-free conditions (perform time-course and dose-response experiments). I believe, it should help. 

Hi, Krzysztof! Thank you so much for all your answers!! I'll definitely try this!! 

Regards, Jordana.

Krzysztof's suggestions are very valuable if TNFalpha is indeed acting "well" in EAhy 926 cells. I am still wondering on this issue; actually some of the papers you mention made me wonder. Please have a look to this paper (Blood paper, free access) in which authors are using pM or 1 nM concentrations of TNF. I mean that these concentrations should be enough to activate its receptor. For instance in the first of the papers you mention, if my calculations are correct, they are using microM concentrations (50 or 100 microg/ml). Anything int he microM range I would think that it is unspecific. In summary I keep wondering whether canonical TNF-mediated signalling is occurring in EAhy 926 cells and/or it may lead to ROS or nitric oxide production.

I totally agree with Krzysztof's suggestions, we also follow exact same things for various endothelial cell stimulations. Normally, in our system, 16-24 hr serum starvation followed by stimulation works really well.