Fluoromax 3 fluorimeter

The stability of curcumin and quercetin content in the positively (CQ_NE+) and negatively charged nanoemulsion (CQ_NE−) stored in refrigerated conditions (2–8 °C) was assessed by determining the two natural compounds’ content in the formulation at predetermined time points by HPLC for up to 30 and 120 days, respectively.
The photostability of curcumin in the same nanoemulsions was determined using curcumin solution in acetonitrile as a control. Briefly, a Hellma^® Ultra Micro Cuvette (Suprasil^® quartz, aperture 1.5 × 5 mm, volume 12 μL; Merck, Darmstadt, Germany) was filled either with a 4 × 10⁻⁴ M curcumin solution in acetonitrile or with CQ_NE− and CQ_NE+ formulations diluted 1:1000 in water and was exposed to 410 nm wavelength light in a Fluoromax-3 fluorimeter (Horiba Jobin Yvon, Palaiseau, France). The cuvette was transparent only in the aperture. This configuration ensured complete irradiation of the solution and excluded diffusion effects due to incomplete irradiation, as the light beam was wider than the aperture. The power of the light transmitted through the aperture of the empty cuvette was 0.36 mW. The photodegradation of curcumin was monitored by absorption spectroscopy. The measurements were performed with a PerkinElmer Lambda 650 UV/vis spectrophotometer (PerkinElmer Italia, Milan, Italy), after 3, 8, 15, 25, and 40 min of exposure, in single ray mode.

TagRFP-T and stagRFP were over-expressed in the JM109 strain using pRSET-B vector at 37 °C then subsequently purified via His-tag affinity chromatography on separate Ni-NTA columns (Qiagen). The proteins were eluted using an imidazole gradient in 20 mM increments up to 200 mM imidazole. Pure fractions were concentrated via Amicon Ultra-15 filters (10,000 NMWL), stored at 4 °C in 50 mM Tris buffer with 300 mM NaCl at pH 7.4, and characterized within one month. Semi-native PAGE of stagRFP was performed using 10% polyacrylamide gels on non-boiled protein samples with minimal SDS content and stained using Coomassie Blue. Purified fluorescent proteins were simultaneously characterized by absorption (Beckman DU-650 spectrophotometer) and fluorescence (Horiba Jobin Yvon FluoroMax-3 fluorimeter). The absorbance spectra were normalized to the respective absorbance at 280 nm and the fluorescence spectra were normalized to the fluorescence maximum of TagRFP-T.

Human amylin (Calbiochem Inc., CA, USA) was dissolved in HFIP to produce a 400 μM stock solution. The stock solution was then added to 40 μM insulin B chain derived peptide (Peptron Inc., Taejeon, Korea) solutions in 10 mM sodium acetate buffer pH 6.5 (10-fold excess for peptides), or to 10 mM sodium acetate buffer alone to achieve a final concentration of 4 μM. Immediately after dilution, the samples were centrifuged for 15 min at 20 K rcf at 4 °C, and the pellet was removed. Aliquots of the reaction solutions were diluted 10-fold in a sodium acetate buffer with 0.03 μM thioflavin T (ThT). Fluorescence values were measured immediately after preparation and after 48 and 73 hours, at an excitation of 450 nm and an emission of 480 nm, using a Jobin Yvon Horiba Fluoromax 3 fluorimeter. The experiment was performed with three independent repeats. The average values are presented, with bars (in the column graph) indicating the standard deviations.