Claran,

What negative controls are you using? Particularly, what unactivated cells?

Since cell activation can occur by exposure to a variety of stimuli, all reagents and conditions used for isolation and growth need to be considered as the source of activation, not just growth substrates. Have you isolated and cultured Fbs from other organs or tissues using similar or identical methodology and reagents? Do these Fbs remain unactivated? Have you evaluated activation just prior to substrate attachment? (This may be difficult, since attachment may be a necessary part of the isolation procedure.)

HI Ciaran,

If you're having trouble with fibroblasts activating in regular culture, it may be worth try a culture model that is designed to induce quiescence. You can try plating the fibroblasts sparsely so they do not touch each other to reduce intercellular signalling (contact-inhibition). You could also try serum-starving the cells.

It's also been found that stiffness of the culture vessel can induce fibroblast activation. Have you tried culturing on growth factor reduced (GFR) matrigel? If you have an you're still having issues, it might be worth looking into another coating material, like GelMA. You can deposit GelMA on the bottom of your culture flask and UV cure it to various degrees of stiffness before seeding your cells. Here's a reference: Article Quiescent Fibroblasts Exhibit High Metabolic Activity

For both these materials, I would recommend checking out OkaSciences. They make really good quality matrigel equivalents and GelMA, and are able to ship next day so the materials are really easy to get! https://okasciences.com/