Cell Signaling Technology
Cyclic AMP XP Assay Kit
4339
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cAMP is an important second messenger in the cell that activates Protein Kinase A. cAMP is generated by the enzymes called adenylate cyclases that are activated by the GalphaS protein. In the present experiment, cAMP levels were measured in 3T3 cells after they were stimulated with Forskolin for 30 min. The goal was to see if 3T3 cells respond to Forskolin and that whether it leads to global increase in cAMP levels.
ELISA
3T3 cell lysates
20,000 cells per well were seeded in a 96 well plate. 48 hr later, confluent cells were serum starved for 20 hr. Cells were then treated with 500 µM IBMX for 30 min. After this treatment period, cells were then treated with three different doses of forskolin in the presence of IBMX for 30 min. Cells were then washed with 1xPBS and lysed, followed by cAMP measurement according to the manufacturer's instructions.
Follow the protocol from the manufacturer
250 nM and 10 µM forskolin treatment resulted in a massive up regulation of cAMP levels.
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