In vitro stimulation of macrophages
Purified Cord Factor was used to stimulate either mouse RAW 264.7 cells or bone marrow-derived macrophages. Cord Factor was suspended at a concentration of 1mg/ml in isopropanol and sonicated in a bath sonicator for 5 min. This suspension was incubated at 60°C for 10 min. and sonication repeated. The resulting solution was layered onto 24-well tissue culture plates at the indicated concentrations and incubated at 37°C in order to ensure complete evaporation of the solvent. Control wells were layered with solvent without Cord Factor and incubated at 37°C. To this layer of Cord Factor, either RAW 264.7 cells or bone marrow-derived macrophages were added at a concentration of 10⁶ cells in 100µl of medium and incubated at 37°C for 24 hours before activation e.g. TNF-α production was measured in the supernatant.
Alternatively, Cord Factor was suspended at a concentration of 0.2 or 2mg/ml in hexane. Of the resulting solution 50µl were layered onto 96-well tissue culture plates at the indicated concentrations of 1 or 10µg/well, respectively and the solvent completely evaporated. Control wells were layered with solvent without Cord Factor and incubated at 37°C. To this layer of Cord Factor, either RAW 264.7 cells or bone marrow-derived macrophages were added at a concentration of 10⁶ cells in 100µl of medium and incubated at 37°C for 24 hours before activation e.g. TNF-α production was measured in the supernatant.

In vivo pulmonary granuloma formation in mice
For in vivo experiments 10µg Cord Factor per mouse were applied i.v. in a water/oil/water emulsion.

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