We opted for dual emission ratiometric FRET measurements supplemented with FRET-fluorescence lifetime imaging microscopy (FLIM) measurements to monitor the kinetics of Gq activation and FRET efficiencies, respectively.
ADE3 was deleted and the growth medium supplemented with adenine to reduce background fluorescence from intermediates of the adenine biosynthesis pathway.
These variants allow characterization of ligand binding and dynamic properties of the enzyme using NMR and fluorescence methods, supplemented with molecular dynamics (MD) simulations.
Briefly, logarithmically growing yeast cells (in liquid low fluorescence medium supplemented with 2% glucose) were exposed to blue light (30 μmol m−2 s−1) for 5.5 hours or kept in darkness for the same amount of time.
Mock-transfected CHO-B2 and α5B2 cells (1×10 cells in 1 ml of DMEM supplemented with 10% FBS) were fluorescence-labelled by 3 μl of 1 m M BCECF-AM DMSO solution (Wako Pure Chemical Industries, Ltd ,Hokkaido, Japan) and incubated for 30 min at room temperature.
Figure 4 Fluorescence of recombinant C. reinhardtii GFP (strain expressing green fluorescent protein) cultivations supplemented with selenocystamine.
Immunofluorescent sections were enclosed in Vinol (Air Products, Allentown, USA) supplemented with DAPI (Invitrogen) and analyzed on a Leica DM6000 fluorescence microscope (Leica Microsystems, Heidelberg, Germany), equipped with LAS AF (Leica) software.
Next, the medium was replaced with RAB buffer (Hepes 10 mM pH 7.4; NaCl 135 mM, KCl 10 mM, MgCl2 0.4 mM; CaCl2 1 mM; Glucose 1%) supplemented with the indicated steroids and then analyzed by confocal fluorescence microscopy.
Addition of thapsigargin in Ca2+-free medium (supplemented with 15 mM EGTA) resulted in an increase of fluorescence in RBCs of T. torquatus loaded with Fluo-3 AM.
For serine starvation experiments in which YFP fluorescence was measured, MSgg medium was supplemented with 800 µM serine and 400 µM serine methyl-ester (Sigma, St . Louis MO).
