Fp 6500 spectrofluorometer

The interaction of α-mangostin with α-glucosidase was studied using the fluorescence quenching method [50 (link)]. The fluorescence of α-glucosidase alone and in the presence of the inhibitor was studied at different concentrations (Jasco FP-6500 spectrofluorometer, Japan). Measurements were made in the emission range of 300–500 nm, with an excitation of 280 nm, after 10 min of stabilization. The fluorescent spectra of α-glucosidase (0.35 µM) and inhibitor (0.05–1.5 µM) were carried out at three different temperatures (298, 304 and 310 K), and the bandwidths were set at 5 nm for both emission and excitation slits. For each sample, three fluorescence spectra were acquired, and the blank was subtracted. The compound quenching mechanism was evaluated using the Stern–Volmer equation [51 (link)]. Synchronous fluorescence spectra were collected in the emission range of 260–320 nm [52 (link)]. The difference between excitation and emission wavelength (Δλ) was established at 15 nm (for tyrosine residues) or 60 nm (for tryptophan residues) [53 (link)].

UV–vis absorption spectra of NBDH, NBD-PD, SBA-NBDH, and SBA-NBD-PD, in solid state/powder, were performed with a Perkin Elmer Lambda 35 spectrometer equipped with an integrating sphere, using a spectral on as a certified reflectance standard, at a scanning speed of 60 nm/min and a slit of 4 nm. Fluorescence emission and excitation spectra of NBDH, NBD-PD, SBA-NBDH, and SBA-NBD-PD, in solid state/powder, were registered using the Jasco FP-6500 spectrofluorometer, equipped with EFA-383 epifluorescence attachment, at a scanning speed of 100 nm/min and an excitation wavelength of 450 nm.

Fluorescence anisotropy was conducted as described previously^{33 (link)}. In brief, 5′-FAM-labelled RNA duplexes (100 nM) were titrated with 0–700 nM ODAGal4 in 10 mM NaH₂PO₄–Na₂HPO₄ buffer, pH 7.0, containing 100 mM of NaCl and 0.02% Tween 20 at 20 °C. The change in fluorescence anisotropy was monitored with a FP-6500 spectrofluorometer (JASCO) by averaging three measurements. The instrument settings were Ex/Em = 490/520 nm; response, 2 s; bandwidth (Ex), 5 nm; bandwidth (Em), 5 nm; PMT voltage, 430 V; number of cycles, 4. The dissociation constant of ODAGal4 and each RNA duplex was calculated by the method of Wang et al.^{69 (link)}

DNA amount and sGAG content in the hydrogel disks were quantified after six weeks of implantation. The harvested cell-laden hydrogel disk implants were washed with PBS three times and freeze-dried. Each of the freeze-dried implants was digested by 500 µL papain solution (Sigma-Aldrich, St. Louis, MO, USA), which was prepared by dissolving papain at a concentration of 400 mg/mL in 0.1 M PBS (pH 6.0) containing 5 mM cysteine hydrochloride and 5 mM ethylenediaminetetraacetic acid (EDTA). 5 µL of the papain digestion solution was used to measure the DNA amount with Hoechst 33258 dye (Sigma-Aldrich, St. Louis, MO, USA). The fluorescence intensity was read with an FP-6500 spectrofluorometer (JASCO, Tokyo, Japan) at an excitation/emission wavelength of 360 and 460 nm. The sGAG content in each digestion solution was measured by using a BlyscanTM Glycosaminoglycan Assay Kit (Biocolor Ltd., County Antrim, UK). Four samples in each group were used for the measurement to calculate means and standard deviations.