480 to 520 nm band pass filter

A detailed description of the isolated and prepared bovine ROS was reported previously (58 (link)). The washed ROS with isotonic and hypotonic buffers was used, in the dark under a dim red light (>670 nm) for observance, to extract rhodopsin as described previously (59 (link), 60 (link), 61 (link), 62 , 63 (link), 64 (link)). Solubilized rhodopsin at 1.05 mg/ml in 20 mM 1,3-bis(tris(hydroxymethyl)methylamino) propane, pH 6.9, 100 mM NaCl, and 1 mM LMNG buffer was incubated for 5 min at 20 °C with 0.7 μl of 100 mM stock solutions of each of the nine identified hit compounds in DMSO (100 μM final concentration) and centrifuged at 1000g for an additional 5 min. Absorption spectra of rhodopsin (dark, and treated with the nine hit compounds and untreated) were then measured with a Cary 50 UV-visible spectrophotometer (Varian). Next, photobleaching was carried out with a 150-W fiber light delivered through a 480 to 520 nm band pass filter (Chroma Technology) for 10 s; immediately then, absorption spectra were measured again for each sample in triplicate.

The washed ROS with isotonic and hypotonic buffers was used as described above. The solubilized rhodopsin at 1.05 mg/ml in 20 mM 1,3-bis(tris(hydroxymethyl)methylamino) propane, pH 6.9, 100 mM NaCl, and 1 mM LMNG buffer was incubated for 5 min at 20 °C with 0.7 μl of 100 mM stock solutions of the nine identified hit compounds (100 μM final concentration) in DMSO and centrifuged at 1000g for an additional 5 min. Next, photobleaching was carried out with a 150-W fiber light delivered through a 480 to 520 nm band pass filter (Chroma Technology) for 10 s. Samples were subjected to fluorescence measurements acquired through 150 s in triplicate. The results were analyzed with an L55 luminescence spectrophotometer (PerkinElmer) operating at excitation and emission wavelengths of 300 nm and 335 nm, respectively.