Promega
cAMP-Glo Assay
V1501
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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.
Bioluminescence assay
3T3 cell lysate
20,000 cells were seed per well in a 96 well plate. Confluent cells were serum starved overnight. The next day, cells were left untreated or treated with 1 µM Forskolin for 30 min. Cells were then washed with 1x PBS. Cell lysis and cAMP measurement was performed according to the manufacturer's instructions.
To enhance cAMP level detection, cells should be pre-treated with phosphodiesterase inhibitors.
3T3 cells respond very well to Forskolin. With 1 µM Forskolin treatment for 30 min, there was a 3 fold increase in cAMP levels.
None
Easy to use
cAMP measurements with this kit is based on inverse relationship of the bioluminescence values. Therefore, it is important that cell number is same across all samples. Otherwise, it can be difficult to normalize values.
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