Sephadex g 25 fine resin

The AGE mixture was incubated (3 h, 37 °C) in 200 µL of an FAOD-containing solution (3.83 U/mL). Gel permeation chromatography of FN3K- and FAOD-treated AGEs was carried out on a column (length: 60 cm, inner diameter: 15 mm) packed with Sephadex G-25^® Fine resin (Sigma-Aldrich), in order to assess the apparent molecular mass of fructose-containing compounds (e.g., AGEs). Following fractionation, the presence of AGEs was first demonstrated based on Maillard-type autofluorescence (AF) measurements (excitation 365 nm, emission 390–700 nm) using a Flame miniature spectrometer (FLAME-S-VIS-NIR-ES, 350–1000 nm, Ocean Optics, Dunedin, FL, USA) equipped with a high-power LED light source (365 nm, Ocean Optics) and a reflection probe (QR400-7-VIS-BX, Ocean Optics). Autofluorescence peaks for FAOD-treated AGEs were detected at a wavelength of 520 nm. All obtained fractions were photometrically measured using the resorcinol–HCl (Seliwanoff) reaction, a classical color reaction for the presence of ketoses [34 (link)]. A 50 µL sample was added to 100 µL resorcinol (9 mM, Sigma-Aldrich) and 1 mL hydrochloric acid (9 mol/L, Sigma-Aldrich). Following a 5 min incubation in a boiling water bath, the color development was read photometrically at 488 nm in a 10 mm cuvette.

Alexa Flour 647, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) was obtained from Invitrogen. Sephadex G-25 fine resin, egg phosphatidylcholine (PC), cholesteryl oleate, sodium cyanoborohydride (NaCNBH₃), triethylamine (TEA), 3-aminopropyl-triethoxysilan (APTES), ethanolamine (ETA), sodium deoxycholate and HEPES were from Sigma. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) was purchased from Avanti Polar Lipids. Cholesterol linked to boron dipyrromethene difluoride at sterol carbon-24 (cholesterol- Bodipy; C-Bodipy) was synthesized as described^{23 (link)}. Cholesteryl-ester- Bodipy (CE-Bodipy) was synthesized by conjugating linoleic acid to C-Bodipy as described²⁴ .