This is something that I've struggled with a lot in different tissue scenarios. The best thing to do, if possible, is a pilot where you test having a transport blocker during cell isolation or incubate the organ or single-cell suspensions in a transport blocker for an extended period of time, and compare directly to restimulated cells. IL1b is pretty highly expressed. You could even try direct staining without block; this often works for some strongly expressed cytokines.

As a backup, I've also used sorting into lysis buffer followed by either qRTPCR or microwesterns.

We found that isolation of cell suspension in the presence of 10 ug/ml of brefelin A was essential for assessing intracellular cytokine expression in cells taken directly from mice. Put the cells in 37 C incubator for at least 4 hours after isolation and then do your intracellular staining procedure. this works for many cytokines. But, IL-1b is unique. It is not a secreted cytokine. It is released from cells when the inflammasome assembles and caspase-1 is activated. You should be able to stain cells for proIL-1beta. There are antibodies available to do this.

Also, a rechallenge will increase your signal, so you should test that as well. Heat-killed bacteria is good. Remember to put in an amendment to your animal protocol if you are going to be challenging mice with heat-killed or live bacteria. Good luck.

I have not looked at IL-1b (proIL-1b), but there are some methods for accessing intracellular cytokine directly ex vivo that may work for your situation.

If you are looking at cells directly from the the BALF, you may try using a golgi-blocker in the media that you use for the lavage and keep it in your media at each step until the cells have been fixed. If you are looking for cellular expression in the lung tissue, traditional enzymatic digestions (collagenase, DNase, liberase, etc.) generally are not very compatible with direct ex vivo ICS (intracellular cytokine staining). However, a quick mechanical digestion in the presence of golgi-blocking chemicals may work.

An additional method for accessing ICS directly ex vivo is by giving your golgi-blocker to the mouse (i.v.) 4-6 hours prior to harvesting the tissue. At harvest, you would follow the same methods as described above (keeping golgi-blocker in media at every step until cells are fixed). This method is in the immunological methods "Gold Book" and was designed for looking at ICS in spleen cells, but can be adapted for tissues. Have fun.

Hi Matthew. Have you tried the in vivo brefeldin A method? If it works, it could be effective.

James. I have done the in vivo BFA method (which is what I was referring to as the method in the Immunology methods "Gold book"). I had limited success with it (looking in the liver at the time), but did not continue pursing it.

I'll be giving the ex vivo ~5h incubation with monensin (rather than brefeldin A...ebiosciences recommends this) after seven days of infection, which I observe to be the peak of IL-1 expression by Western blot/ELISA (~4000pg in 1mL lung homogenate). If that doesn't go very well I'll try restimulating with heat-killed bacteria and let you all know how it goes. Thanks for the advice!