I want to check if a batch of TNF-alpha is still biologically active and I am wondering if anyone knows the best way to do that? What readout after stimulation of cells with the TNF would give the most accurate quantification of the activity of that TNF?
IF you have access to endothelial cells or fibroblasts, you can do a 4-6 hr stimulation (with 10 ng/mL TNF) and check for ICAM-1 upregulation by western blotting.
For a quicker response, you can also do a 30 minute stimulation and look for p65 phosphorylation and/or nuclear translocation.
These are all well established and consistent measures for detecting TNF induced inflammatory responses.
Good luck!
Thank you for the help. I will get some endothelial cells and try this technique.
Thank you.
In addition to the above answers a simple functional assay (e.g. neutrophil or monocyte adhesion to EC) may be a good and simple way to check TNF-a activity. In this case you wouldn't need to analyze for any transcription factor(s) or specific adhesion protein(s) (e.g. ICAM-1, VCAM-1 etc) which would be induced by TNF-a. The net TNF-a-induced activation of EC would increase pro-adhesive phenotype resulting in a marked adhesion of leukocytes to EC. This could be simply evaluated under the phase contrast microscope.
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