Drugs first and then cytokines, or at same time? - (Sep/30/2009 )
I am investigating the immunosuppressive/anti-inflammatory effects of novel drugs using the Caco-2 cell line. The drugs are envisioned as novel therapeutics for Crohn's disease and Ulcerative colitis. We add TNFa and IL-1b to the cell media to induce the production of pro-inflammatory markers, such as MMP-9.
For a few months I have been adding the cytokines and drugs simultaenously. The drugs havent been reducing MMP-9 or other markers, even though I know that these two specific drugs worked prior to me starting this project. My supervisor has advised that I should be treating the cells with the drug, waiting an hour, and then adding the cytokines.
The problem I have here is that this doesnt make sense from a therapy viewpoint. If the drugs are only effect at blocking the initial pro-inflammatory signal but cant reduce an established signal, then should I be performing the experiment in the way advised by my supervisor?
welcome to the research world of immunosuppression, where some labs add stimulant and immunosuppressant at the same time, some add the immunosuppressive agent first (1-12h before) and some add after the stimulant (30-12h).
I too have asked ... why add the anti-inflammatory agent prior to the immune stimulant since this is not the order that occurs in the body. The answer ... it is the way it is done.
CellSpecific is also right ... do as the PI says.
My solution ... if not too hard, try all variations and see what you get.
Also, are you looking for an RNA response or protein response? How long is your treatment time? This could also be a factor as protein may need 6-24h treatment before you can measure a response, and some genes are "slow".
Sorry for not being able to give a concrete answer but enjoy.