Fp 6300

The FAST method (Fluorescence of Advanced Maillard products and Soluble Tryptophan) has been used in previous works to discriminate heat-treatments in milk [56 (link),57 (link)]. However, it was found that the degree of denaturation of whey proteins increases during frozen storage [58 ].
Mozzarella cheese sample (0.5 g) was mixed to 4.5 mL sodium acetate buffer (0.1 M, pH 4.6) and shanked vigorously for 30 s. After centrifugation at 4000× g for 10 min at room temperature, the supernatant was diluted (1:10), filtered through a 0.45 µm filter, and analyzed by Spectrofluorometer (FP-6300, Jasco).
Two fluorescence intensities were measured:

Fluorescence of tryptophan at 290 nm excitation and 340 nm emission (Trp-F)

Fluorescence of advanced Maillard’s products at 350 nm excitation and of 440 nm emission (AMP-F) and expressed in arbitrary units (a.u)

The FAST index was calculated as follows:
In acidic conditions (pH 4.6), caseins precipitate, and some whey proteins also precipitate if denatured by heat or freezing. Trp-F measurement is also an estimate of the content of soluble whey proteins present in the supernatant and therefore of the undenatured proteins.
Samples containing more denatured protein are characterized by lower Trp-F values and higher FAST indexes. When advanced Maillard’s reaction occurs, samples present higher AMP-F values and higher FAST indexes.

Quantitative GUS fluorometric assays were conducted according to the method of Jafferson [40 (link)] by measuring the fluorescence of 4-methylumbelliferone (MU) formed as a result of the cleavage of 4-methylumbelliferyl-β-D-glucuronide (MUG, Himedia) by GUS with a spectrofluorometer FP-6300 (JASCO, Japan). GUS activity was calculated as the production of MU from MUG in pmol/min/μg of protein.

DHODH activity in cell lysates were measured following the previous report.³³ A total of 300 μL cell lysate was mixed with in an aqueous solution (total volume, 1.0 mL) containing 500 μmol·L⁻¹ DHO, 200 mmol·L⁻¹ K₂CO₃-HCI (pH 8.0), 0.2% triton X-100, and 100 μmol·L⁻¹ coenzyme Q10 at 37 °C 30 min. An aliquot (100 μL) of the mixture of enzyme reaction mixture was mixed with 100 μL of 0.5 μmol·L⁻¹orotic acid, 50 μL of H₂O, 250 μL of 4.0 mmol·L⁻¹ 4-TFMBAO, 250 μL of 8.0 mmol·L⁻¹ K₃[Fe(CN)₆], and 250 μL of 80 mmol·L⁻¹ K₂CO₃ and then heated at 80 °C for 4.0 min. The reaction was stopped by cooling in an ice-water bath and the FL intensity was measured with a spectrofluorometer (FP-6300 Jasco, Tokyo, Japan): excitation and emission wavelengths were 340 nm and 460 nm, respectively.

H₂O₂ production from SH-SY5Y cells during in vitro incubation was measured using the H₂O₂-specific fluorescent dye Amplex Red (N-acetyl-3,7-dihydroxyphenoxazine) as described earlier [11 , 29 (link)]. Briefly, control and treated cells were harvested and washed twice with phosphate-buffered saline (PBS) and finally resuspended in Krebs-Ringer's buffer containing 10 mM glucose (pH 7.4). An aliquot of this cell suspension was incubated in the same Krebs-Ringer's buffer with 50 μM Amplex Red and 1 U/mL horseradish peroxidase for 15 min in the dark at room temperature. At the end of the incubation an aliquot of the incubation mixture was appropriately diluted and the fluorescence emission was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm using a spectrofluorometer (FP6300, JASCO International Co., Japan). The background fluorescence was subtracted from the sample fluorescence using a blank containing the reaction mixture without any added cells. Fluorescence intensity was converted to nmol of H₂O₂ produced per mg of protein using a calibration curve utilizing pure H₂O₂ in the concentration range of 50 nM to 400 nM.

Mitochondrial transmembrane potential was measured in intact SH-SY5Y cells by using a cationic carbocyanine dye JC-1 which remains distributed in the cytosol as monomers while after entering the mitochondria driven by the electrochemical gradient undergoes concentration-dependent aggregation to form J-aggregates. The monomers emit a green fluorescence (λex 490 nm, λem 530 nm) and J-aggregates a red fluorescence (λex 490 nm, λem 590 nm) and the ratio of fluorescence intensities at 590 nm to 530 nm indicates mitochondrial transmembrane potential [11 ]. Briefly, control and treated cells were washed twice in PBS and then incubated in serum-free DMEM for 30 min in the presence of JC-1 (10 μM) at 37°C in the dark. The cells were pelleted down and then washed thrice with serum-free DMEM, suspended with suitable dilutions in the same medium and the fluorescence emission intensities (590 nm and 530 nm) were measured with excitation at 490 nm in a spectrofluorometer (FP-6300, JASCO International Co., Japan).

The mean distance between DNA-PEG-lipids within PMs was calculated from the concentration of unbound DNA-PEG-lipids. First, fluorescence spectra of HBS solutions containing FITC-labeled DNA-PEG-lipids with known concentrations (0.5–2 μg/ml) were obtained by using a fluorescence spectrometer (FP-6300, JASCO, Japan) [Fig. S8(A)]. The excitation wavelength was 495 nm. Then, a calibration curve was obtained by plotting the fluorescence intensities at 520 nm as a function of the concentration of DNA-PEG-lipids [Fig. S8(B)]. Next, PMs with E-cadherin were prepared in a PDMS chamber with a hollow diameter of 20 mm, which is larger than those used in the cell experiments. The PM was incubated with 1 ml of a solution containing 1.5 μg/ml FITC-labeled DNA-PEG-lipids for 30 min at 37 °C. The solution was then diluted to 2 ml. After gentle pipetting, 1 ml of the solution was collected, and the fluorescence spectrum was measured by using a fluorescence spectrometer. The concentration of unbound DNA-PEG-lipids was calculated from the calibration curve. Finally, by taking the molecular weight of DNA-PEG-lipids (5000) and the known chamber area, a mean distance between DNA-PEG-lipids ⟨d_DNA⟩ could be determined. We conducted three independent experiments to determine the mean distance.