Fluorescent Imaging of cGMP in Cells

FRET/cGMP imaging of cells ex vivo was performed in flow chambers (ibidi) using an epifluorescence setup (Supplementary Fig. 3a) based on an inverted Axiovert 200 microscope (Zeiss) equipped with EC Plan NeoFluar 10×/0.3, LD Plan NeoFluar 20×/0.4 air, and Plan NeoFluar 40×/1.3 oil objectives and optional 1.6× Optovar magnification (Zeiss). The imaging setup contains a light source with excitation filter switching device (Oligochrome, TILL Photonics GmbH), a DualView beam splitter with 516 nm dichroic mirror and CFP and YFP emission filters (480/30 nm and 535/40 nm) (Photometrics), and a CCD digital camera (Retiga 2000R, QImaging)^{22 (link)}. Images were acquired at 0.2 Hz or 1 Hz at room temperature. Adherent cells were exposed to flow at a shear rate of 500 s⁻¹ using a syringe pump (B-Braun). Platelet thrombi and VSMCs were superfused at room temperature with platelet Tyrode buffer and imaging buffer (in mM: 5 HEPES, 140 NaCl, 5 KCl, 1.2 MgCl₂, 2.5 CaCl₂, 5 d-glucose, pH 7.4), respectively. Drugs were applied via two sample loops connected in series and controlled by injection valves (Pharmacia V-7, GE Healthcare).