Fp 6200

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

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.

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).

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)].

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%.