Ratiometric FRET measurements were performed using a previously described wide-field fluorescence microscope21 (link). Typical exposure times ranged from 50 ms to 150 ms, and camera binning was set to 4 × 4. Fluorophores were excited with 420 nm light (slit width 30 nm) and reflected onto the sample by a 455DCLP dichroic mirror and CFP emission was detected with a BP470/30 filter, and YFP emission was detected with a BP535/30 filter by rotating the filter wheel. All acquisitions were corrected for background signal and bleedthrough of CFP emission in the YFP channel (around 55% of the intensity measured in the CFP channel).
In dynamic experiments, cells were stimulated with 100 μM histamine (Sigma-Aldrich) and 10 μM Mepyramine (Sigma-Aldrich) or 100 nM Rapamycin (LC Laboratories, Woburn, USA). Where indicated, cells were incubated with 100 ng/ml Pertussis Toxin (PTX) (Sigma Aldrich) overnight. The Gαq inhibitor FR90035935 was added to cells 2 hours before the measurements started at a concentration of 2 μM and was purchased from the University of Bonn (http://www.pharmbio.uni-bonn.de/signaltransduktion ).
In dynamic experiments, cells were stimulated with 100 μM histamine (Sigma-Aldrich) and 10 μM Mepyramine (Sigma-Aldrich) or 100 nM Rapamycin (LC Laboratories, Woburn, USA). Where indicated, cells were incubated with 100 ng/ml Pertussis Toxin (PTX) (Sigma Aldrich) overnight. The Gαq inhibitor FR90035935 was added to cells 2 hours before the measurements started at a concentration of 2 μM and was purchased from the University of Bonn (
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