cAMP ELISA

Cells were grown in 10 cm dishes to confluence and split 1:15 into 60 mm dishes and grown to confluence. Complete growth media was exchanged for serum-free media and cells were allowed to incubate overnight. Cells were either untreated or treated with 10 μM IBMX for 2 min at room temperature under red safety lighting. Cells that were not treated with IBMX were also exposed to red safety lighting for 2 min prior to further manipulation. After the 2 min incubation, media was aspirated, cells exposed to 10 s or 20 min 440 nm LED light, and allowed to incubate in the dark for an additional 10 s before lysis with 500 μL 0.1 M HCl + 0.05 % Triton-X 100 (Sigma). Dark samples were treated exactly the same, except they were not exposed to 440 nm light. All samples were allowed to lyse in the dark for at least 10 min at room temperature. Each plate was tapped to lift cells and lysates were transferred to 1.5 mL centrifuge tubes and vortexed 5 – 10 s at maximum speed before being centrifuged at 10,000 × g for 10 min at 4 °C. Clarified supernatants were used for cAMP Direct ELISA (Enzo). 100 μL each sample was used per well. Each sample was run in duplicate. 20 μL of each lysate was used for protein content measurement by the Pierce 660 nm protein assay. The cAMP Direct ELISA was run in its acetylated format according to manufacturer’s protocol (see: http://www.enzolifesciences.com/fileadmin/files/manual/ADI-900–066_insert.pdf).