Peripheral blood mononuclear cells (PBMCs) were incubated overnight at 50,000 to 100,000 cells/well in 96-well polyvinylidene plates that had been precoated with 0.5 μg/ml anti-human interferon-γ monoclonal antibody (Mabtech, Stockholm, Sweden).

Following injection, cells were incubated overnight at 37 C and patch clamp experiments were performed the following day.

Plates were incubated overnight at 37 C and stored at -80 C. To prepare template plasmid DNA from each sample, bacterial inoculates were transferred from 384-well storage to 96-well growth blocks containing 1 ml medium per sample and grown overnight.

A loopful of cells was inoculated into 500 μl of sterile water and the mixture was incubated overnight at 28 C with 10 μl of β-glucuronidase (Sigma G7770).

All tubes were mixed, incubated overnight at 4 C.

Cell lysates were incubated with 2 μg of rabbit polyclonal antibody overnight at 4 C on a rotator.

For imaging studies, MDCK cells were plated on glass-bottomed 35 mm culture dishes (MatTek) at 5 x 10 3cells/cm 2in growth medium and incubated overnight, transfected with the relevant plasmid(s), changed into serum-free medium to quiesce the cells, incubated overnight, and used the next day.

Sections were incubated overnight at 4 C. Immunoreactivity of the primary antibody was then labeled either with the chromogen diamino benzidine (DAKO Diagnostika, Hamburg) or with a fluorophore-coupled secondary antibody.

The ligation reaction was incubated overnight at 14 C.

After a 3-hour incubation, a nutrient rich overlay gel was poured and the plates were incubated overnight.