Hi Yara, it is always a good idea to add proteinase inhibitor cocktail to biological samples kept at room temp.

In general, all proteinases are inactive at -80oC. (they are hydrolases that require free H2O and at -80oC, there is almost no free H2O). 

Some are active at -20oC, but all are active at +4oC to +25oC.

Thank you for your answer.

Do you think that proteases could be active the moment I thaw the samples, and during diluting and manipulating them before starting the protocol on the plate?

I tried the the Multiplex assay without adding Protease cocktail inhibitor to BAL and I detected nothing in the samples (control and exposed groups of mice), do you think the fact that I didn't add the Protease inhibitors has influenced the results?

Hi Yara, what do you mean by:

"I tried the the Multiplex assay without adding Protease cocktail inhibitor to BAL and I detected nothing in the samples (control and exposed groups of mice), "?

Do you mean that you did not detect any cytokines in the samples without added proteinase inhibitors?

Have you tried thawing samples in the presence of proteinase inhibitors?

I'm asking this because many outside/inside interfaces in mammals like lungs, stomach, intestines, etc are heavily guarded by inflammatory cells. The nastiest of the lot are the neutrophils which contain large loads of proteinases that can quickly chew up almost all proteins. 

I mean that there was no protease cocktail inhibitor in my samples and I was wondering if this would be the reason behind this result (all the cytokines weren't detected).

Do you think centrifuging BAL directly to remove cells (macrophages and other white blood cells) and adding anti proteases directly would solve the problem?

Thank you!

This is a tricky subject because inflammatory cells contain cytokines, cytokine precursors and proteinases that activate or inactivate cytokines.

Personally, I would include proteinase inhibitor cocktails in everything, just to exclude that variability.