For the last few experiments I am getting no elevation in ROS comparing to the control group (No TNF-a). Generally I used to get ~30% increase (Amplex Red Assay. I am keeping the cells starved for 24hr then after a single wash with PBS applying TNF-a (prepared in EBM-2, final con. 10ng/ml).
So, if I need to check whether my TNF-a stock is functional, what should I do?
Thanks in advance.
A positive control experiment,which has been reported in the articles, may be helpful.
Hello !
The 30% increase following TNF-a treatment seems very low. What kind of cells do you use ? Maybe you can try another cell type. TNF-a effect is not restricted to H2O2 release. If applicable, you can measure cytokines production or genes expression to check TNF-a activity.
Dear Suzanne
Thank you for answering. I am using HUVEC (passage 5-10). Sometimes in TNF-a treated group I did get values lower than that of control group. That really puzzled me.
If you have anymore suggestion, please share.
Do you use many different batches of HUVEC ? Have you checked NOX expression in HUVEC ? Which one is the mainly expressed ? Have you tried to measure superoxyde anion release ?
Maybe, you can also assess adhesion molécules expression upon TNF-a treatment.
Hope it will help you...
Dear Suzanne,
Frankly speaking, due to some economic issue I had to use different batches, and for the same reason I couldn't perform the others.
I found in many articles that they got almost a 3 fold increase in ROS comparing to the control, but unfortunately there were scarcity of information like, starvation time, percentage of FBS, what was actually the control.... so as a beginner it was hard for me to design an accurate procedure to generate and measure the ROS. Until now I have been looking for a complete protocol.
I will certainly go for the VCAM-1, ICAM-1 assay.
Thank you again.
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