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65 protocols using fp 6200

1

Quantification of Bone Collagen Crosslinks

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The analysis of collagen crosslinks in bone was performed according to the procedure reported previously [27 (link)]. Briefly, bone powder of the femoral shaft after biomechanical testing was prepared and demineralized twice with 0.5 M EDTA in 50 mM Tris buffer (pH 7.4) for 96 h at 4 °C. The demineralized bone residues were then suspended in 0.15 M potassium phosphate buffer (pH 7.6) and reduced at 37 °C with NaBH4. The reduced specimens were hydrolyzed in 6N HCl at 110 °C for 24 h. Hydrolysates were analyzed for the content of crosslinks and hydroxyproline on a Shimadzu LC9 HPLC fitted with a cation exchange column (0.9 × 10 cm, Aa pack-Na; JASCO, Ltd., Tokyo, Japan) linked to an inline fluorescence flow monitor (RF10AXL; Shimadzu, Shizuoka, Japan). The total content of AGEs was measured by the method of Saito et al. [20 (link)]. Briefly, AGE content was determined using a fluorescence reader at 370 nm excitation and 440 nm emission (JASCO FP6200; JASCO) and normalized to a quinine sulfate standard.
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2

Photocatalytic Degradation of Methylene Blue

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The photocatalytic activity of the prepared anodic oxide was investigated via the decomposition of methylene blue (MB) under UV illumination. UV light in the range 340–400 nm with a central wavelength of 365 nm was supplied using a xenon lamp (MAX-35, Asahi Spectra, Tokyo, Japan). The intensity of the radiated light was 1.0 mW/cm2 at the surface of the cell comprising the MB solution (3.19 ppm) and anodized alloy. The photocatalytic activity was evaluated by measuring the absorbance of MB at 664 nm using a UV-vis spectrophotometer (Jasco V-550, JASCO Corporation, Tokyo, Japan) [25 (link),30 (link)]. The photogenerated hydroxyl radicals (•OH) react with terephthalic acid (TA) in a disodium terephthalate (NaTA) solution, thereby producing 2-hydroxyterephthalic acid (HTA) via a hydroxylation reaction, as shown in the following Equation (1):
The excited light at a wavelength of 315 nm generates HTA and emits fluorescence at a wavelength of 425 nm [31 (link)]. A quartz cell, containing 2 mL of TA (2 mM) and anodized oxide, was illuminated using a xenon lamp at an intensity of 1.0 mW/cm2, and the fluorescence intensity was measured using a spectrometer (Jasco FP-6200, JASCO Corporation, Japan).
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3

Intrinsic Fluorescence Spectroscopy of Protein

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The intrinsic
spectra of the ectodomain G protein at three different pH values were
recorded by a Jasco spectrofluorometer (FP6200) at 25 ± 1 °C
with a cell of a path length 1.0 cm quartz cuvette, and the temperature
of the protein sample was controlled by a circulating water bath.
We monitored the changes in the fluorescence intensity of the protein
with different buffers taking the entrance and exit slit width of
5 nm. The emission spectra of the protein were observed in the wavelength
range of 300–400, and the excitation wavelength was set at
280 nm. All of the spectra at a particular pH value were recorded
in triplicate using the protein concentration of 0.3 mg mL–1.
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4

Oxidative Stress and Apoptosis Evaluation in Biological Samples

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The level of malondialdehyde (MDA), a marker of lipid peroxide, was estimated using the method of Buege and Aust.17 (link) Caspase-3 was evaluated using the Caspase-3/CPP32 Fluorometric Assay Kit (K105) purchased from Biovision, Inc (Mountain View, California). For each assay, Samples were read in a fluorimeter equipped (Jasco, FP-6200; Jasco, Japan) with a 400-nm excitation and a 505-nm emission filter. Fold-increase in Caspase-3 activity was determined by comparing fluorescence of 7-amino-4-trifluoromethyl coumarin in control and treated animals. CYP4502E12E1 activity (p-nitrophenol hydroxylase activity) was determined in the liver tissue through the measurement of p-nitrocatechol generated by an enzymatic reaction using high-performance liquid chromatography method.18 (link)
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5

Spectrofluorometric Analysis with Jasco FP-6200

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Spectrofluorometric apparatus, Jasco FP-6200, Tokyo, Japan.
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6

Measurement of Cytosolic and Mitochondrial Calcium

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Cytosolic and mitochondrial Ca2+ concentration was measured by using, respectively, the fluorescent indicator Fluo-4 AM and X-Rhod-1AM (Invitrogen, Carlsbad, CA, USA). CTRL and Pt primary fibroblasts were grown in a T25 Flask. Cells at 80% confluence were incubated with a fluorescent probe for 30 min at 37 °C. Cell monolayers collected by trypsinization and centrifugation were resuspended in a buffer containing 10 mM HEPES and 6 mM d-Glucose (pH 7.4) at an approximate concentration of 1 × 105 cells in 1 mL. Fluorescence intensity was measured at 25 °C in a spectrofluorometer (Jasco FP6200 Mary’s Court Easton, MD, USA), equipped with a stirrer and temperature control, by the subsequent addition of 5 mM CaCl2, 0,1% Triton X-100 (for cytosolic Ca2+ levels), 0.1% Na-Cholate (for mitochondrial Ca2+ levels) and 40 mM EGTA. The excitation/emission wavelengths were 495 nm/506 nm for Fluo-4 AM and 580 nm/602 nm for X-Rhod-1 AM. The cytosolic and mitochondrial Ca2+ levels were evaluated by using an apparent Kd (443 nM for Fluo-4AM and 700 nM for X-Rhod-1AM) according to the equation described by Grynkiewicz et al. [42 (link)]. Where indicated, incubation with 1 µM Thapsigargin, 10 µM Dantrolene, and 5 µM Ruthenium Red (RR) was performed for 30 min at 37 °C.
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7

Stearyl-r8 Peptide Attachment to EVs

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Synthesized stearyl-r8 peptide (final 40 μM) diluted with phosphate-buffered saline (PBS) was added to a solution of EVs (100 μg) in PBS (total 800 μl) and incubated for 1 h at 37 °C. Removal of unattached stearyl-r8 peptide was accomplished by washing with PBS and filtration using Amicon Ultra centrifugal filters (100K device, Merck Millipore). The attachment of stearyl-r8-GC(Alexa488) and r8-GC(Alexa488) to EVs was confirmed using a spectrofluorometer (FP-6200, JASCO, Tokyo, Japan).
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8

Glycation of BSA by Fructose

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Glycated BSA was prepared using treatment of BSA with different concentration of fructose (200 and 500 mM) at different time periods (1, 2, 3, and 4 weeks) [14 (link)]. Aminoguanidine (AG) a known antiglycation agent was used as a positive control. After dialysis in PBS, glycated BSA formation was determined using a fluorometry method at an excitation wavelength of 440 nm and emission wavelength of 460 nm (spectrofluorometer, Jasco FP-6200) [14 (link)]. Nitroblue tetrazolium (NBT) reaction was used to measure the fructosamine level [15 (link)].
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9

Encapsulation of Fluorescent Cargo in EVs

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To load the fluorescently labeled dextran into EVs, EVs (25 μg) were mixed with FITC-labeled dextran (molecular weight: 70,000) (Sigma-Aldrich) or saporin (50 μg) in PBS (100 μl). After electroporation (poring pulse: two pulses (200 V, 5 msec); transfer pulse: five pulses (20 V, 50 msec)) in a 1-cm electroporation cuvette at room temperature using a super electroporater NEPA21 Type II (NEPA Genes, Tokyo, Japan), the unencapsulated FITC-dextran or saporin was removed by washing and filtration using Amicon Ultra centrifugal filters (100 K device), as previously reported9 (link)10 (link). Loading of FITC-dextran and FITC-saporin into EVs was confirmed using a spectrofluorometer (FP-6200, JASCO, Tokyo, Japan)9 (link)10 (link), and the loading concentration was calculated on the basis of the calibration curve of FITC (excitation: 488 nm; emission: 530 nm). The electroporation method resulted in the encapsulation of FITC-dextran (5 ng/ml) in 1 μg/ml of EVs. The efficiency of dextran encapsulation into EVs was calculated to be 0.3%. The concentration of saporin encapsulated in 10 μg/ml EVs was estimated to be approximately 43.5 ng/ml using the FITC-labeled saporin. The efficiency of saporin encapsulation into EVs was calculated to be 0.2%.
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10

Spectrofluorometric Quantification of Bile PAH Metabolites

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The concentration of the bile PAH metabolite hydroxypyrene was determined by semi-quantitative analysis of the metabolites fluorescence (Aas et al. 2000) . It consisted in measuring the fluorescence by a spectrophotometer with a 5-nm slit width on emission and excitation channels (Jasco FP-6200). Analyses were performed using excitation-emission wavelengths: 343 to 383 nm (4-ringed compounds including pyrene-type metabolites) (Aas et al. 2000) .
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