I would like to graph the trend of increasing intracelluler cAMP after treatment of GLP-1 by concentration in Pancreatic cell (INS-1, RIN-m5F). However, I'm measuring the cAMP using the current kit (R & D system, KGE002B), but I could not confirm the tendency to increase. The kit was a competitive assay and the method proceeded as follows.
Method
RIN-m5F cell 96 well seed to 1*10^5 cells/well/100uL. And incubaiton 37'C CO2 incubator 24hr after day, glucose/FBS without media stavated 2hr in CO2 incubator. Remove the media and wash with the PBS GLP-1 samples were serial diluted 10-fold from 1000 nM. (Dilution buffer: 500 uM IBMX and 100 uM RO-1724 with PBS) GLP-1 samples were loaded in 100 μl / well and reacted for 20 min at RT. The solution was removed and lysed with cell lysis buffer for 10 min. The mouse monoclonal cAMP Ab was coated on the Goat anti-mouse Ab pre-coated plate. After washing plate with PBST, cAMP-HRP and GLP-1 sample (lysis) were mixed with 50 uL and 100 uL / well and incubated for 2 hours at RT. After washing with PBST, TMB substrate was dispensed.
Why is cAMP not dependent on concentration? I would like to receive advice on the method. Is there a best reference for plotting cAMP concentration-dependently in pancreatic cells (INS-1, RIN-m5F)?
There are standard commercial ELISA kits for cAMP measurement. The issue is rapid and complete lysis of the cells. Pancreatic cells should be pretty easy to freeze-thaw, or you could use the Barocycler (Pressure Biosystems) for more consistent recoveries, if you have access to one. Failing those, bead-beating may be your best choice. If you need to know in vivo cAMP levels, your best bet is to do some sort of cAMP-triggered genetic regulation system. Unfortunately those are typically off-on, not really set up for concentration-dependence.
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