Simultaneous optical measurements of cytosolic Ca2+ and cAMP in single cells.
Understanding the temporal and spatial integration of the Ca2+ and adenosine 3',5'-monophosphate (cAMP) signaling pathways requires concurrent measurements of both second messengers. Here, we describe an optical technique to simultaneously image cAMP and Ca2+ concentration gradients in MIN6 mouse insulinoma cells using Epac1-camps, a Förster (or fluorescence) resonance energy transfer (FRET)-based cAMP biosensor, and Fura-2, a fluorescent indicator of Ca2+. This real-time imaging method allows investigation of the dynamic organization and integration of multiple levels of signal processing in single living cells.
Harbeck
Mark C
MC
Department of Medicine, The University of Chicago, Chicago, IL 60637, USA.
Chepurny
Oleg
O
Nikolaev
Viacheslav O
VO
Lohse
Martin J
MJ
Holz
George G
GG
Roe
Michael W
MW
D000818:Animals; D015374:Biosensing Techniques; D002118:Calcium / Q000032:analysis*; D002413:Cations, Divalent; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000032:analysis*; D003600:Cytosol / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes; D016257:Fura-2; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis; D015203:Reproducibility of Results; D015290:Second Messenger Systems; D014162:Transfection
D002413:Cations, Divalent; D005456:Fluorescent Dyes; D020662:Guanine Nucleotide Exchange Factors; D011994:Recombinant Proteins; D000242:Cyclic AMP; D002118:Calcium; D016257:Fura-2