Cellular cAMP accumulation assays

sAC KO MEFs or 4-4 cells (2.0 × 10⁶ cells/ml) in suspension were transferred to 1.5 ml tubes and incubated at 37°C, 5% CO₂ for one hour. A time zero value for each condition was determined by adding 100 µl of cells directly into 100 l stop solution (0.2 M HCl). To measure cAMP accumulation, cells in suspension were incubated for the indicated period of time in the presence of 500 M IBMX (± LRE1 or ± KH7) at 37°C after which 100 l of cells were transferred to a fresh tube containing stop solution. Intracellular cAMP content was determined using Correlate-EIA Direct Assay (Assay Designs, Inc). INS-1E insulinoma cells were incubated in 2.5 mM glucose Krebs-Ringer buffer (pH 7.5) supplemented with 2 mM sodium bicarbonate, 10 mM HEPES, and 0.1% BSA for 2 h before start of the experiment. At time zero for each experiment, media was switched to Krebs-Ringer buffer containing 2.5 mM glucose or 16 mM glucose in the presence of 500 µM IBMX and inhibitor at the shown concentrations. After 10 min cells were lysed in 200 µl 0.1 M HCl. Intracellular cAMP contents were determined using Correlate-EIA Direct Assay (Assay Designs, Inc). Accumulated cAMP quantitated after 10 minutes are presented as pmol cAMP accumulated per 2.5 × 10⁵ cells.