Figure 1: cAMP Standard was diluted in 1X Cell Lysis Buffer #9803 and samples were assayed following the Cyclic AMP XP ® Assay Kit protocol. This standard curve is for demonstration purposes only; users should generate a standard curve for each sample set in order to accurately determine cAMP concentration.
![Figure 2: Treatment of CHO cells with Forskolin #3828 increases cAMP concentration as detected by Cyclic AMP XP ® Assay Kit #4339. CHO cells were seeded at 4x10 4 cells/well in a 96-well plate and incubated overnight. Cells were either left untreated or pretreated with 0.5 mM IBMX for 30 minutes prior to forskolin treatment (15 minutes) and lysed with 1X Cell Lysis Buffer #9803. The absorbance values (left) and percentage of activity (right) are shown above. The percentage of activity is calculated as follows: % activity=100x[(A-A basal )/(A max -A basal )], where A is the sample absorbance, A max is the absorbance at maximum stimulation (i.e., high forskolin concentration), and A basal is the absorbance at basal level (no forskolin). Forskolin directly activates adenylyl cyclases and increases cellular cAMP concentration. IBMX is a non-specific inhibitor of cAMP and cGMP phosphodiesterases and promotes accumulation of cAMP and cGMP in cells.](http://www.bioprodhub.com/system/product_images/ak_products/1/sub_1/1762_4339_graph_fig2.jpg)
Figure 2: Treatment of CHO cells with Forskolin #3828 increases cAMP concentration as detected by Cyclic AMP XP ® Assay Kit #4339. CHO cells were seeded at 4x10 4 cells/well in a 96-well plate and incubated overnight. Cells were either left untreated or pretreated with 0.5 mM IBMX for 30 minutes prior to forskolin treatment (15 minutes) and lysed with 1X Cell Lysis Buffer #9803. The absorbance values (left) and percentage of activity (right) are shown above. The percentage of activity is calculated as follows: % activity=100x[(A-A basal )/(A max -A basal )], where A is the sample absorbance, A max is the absorbance at maximum stimulation (i.e., high forskolin concentration), and A basal is the absorbance at basal level (no forskolin). Forskolin directly activates adenylyl cyclases and increases cellular cAMP concentration. IBMX is a non-specific inhibitor of cAMP and cGMP phosphodiesterases and promotes accumulation of cAMP and cGMP in cells.
![Figure 3: Treatment of 293 cells with isoproterenol increases the cAMP concentration as detected by Cyclic AMP XP ® Assay Kit #4339. 293 cells were seeded at 3x10 4 cells/well in a 96-well plate and incubated overnight. Cells were pretreated with 0.5 mM IBMX for 30 minutes prior to isoproterenol treatment (3 minutes) and lysed with 1X Cell Lysis Buffer #9803. The absorbance values (left) and percentage of activity (right) are shown above. The percentage of activity is calculated as follow: % activity=100*[(A-A basal )/(A max -A basal )], where A is the absorbance of the sample, A max is the absorbance at maximum stimulation (i.e., high isoproterenol concentration), and A basal is the absorbance at basal level (no isoproterenol). Isopropterenol is a β-adrenoceptor agonist and activates beta-2 adrenergic receptors (ADRB2) that are endogenously expressed on 293 cells. Activation of ADRB2 then leads to activation of adenylyl cyclase and synthesis of cAMP as its second messenger.](http://www.bioprodhub.com/system/product_images/ak_products/1/sub_1/1763_4339_graph_fig3.jpg)
Figure 3: Treatment of 293 cells with isoproterenol increases the cAMP concentration as detected by Cyclic AMP XP ® Assay Kit #4339. 293 cells were seeded at 3x10 4 cells/well in a 96-well plate and incubated overnight. Cells were pretreated with 0.5 mM IBMX for 30 minutes prior to isoproterenol treatment (3 minutes) and lysed with 1X Cell Lysis Buffer #9803. The absorbance values (left) and percentage of activity (right) are shown above. The percentage of activity is calculated as follow: % activity=100*[(A-A basal )/(A max -A basal )], where A is the absorbance of the sample, A max is the absorbance at maximum stimulation (i.e., high isoproterenol concentration), and A basal is the absorbance at basal level (no isoproterenol). Isopropterenol is a β-adrenoceptor agonist and activates beta-2 adrenergic receptors (ADRB2) that are endogenously expressed on 293 cells. Activation of ADRB2 then leads to activation of adenylyl cyclase and synthesis of cAMP as its second messenger.