Spectrofluorometer

Actin polymerization rates were determined by the increase in fluorescence caused by the incorporation of pyrene-labeled actin into actin filaments [34 (link),35 (link)]. Pyrene-labeled actin was pre-cleared by dialysis against G-buffer (10 mM Tris-HCl, pH 8.0, 0.2 mM CaCl₂, 7 mM β-mercaptoethanol, 1 mM ATP) and centrifugation at 100,000× g for 30 min. In these tests, we used pyrene-labeled skeletal muscle actin that was added to the c-actins at a ratio of 20:1 (0.25% to 5% of the c-α-actin). Since pyrene-labeled skeletal-actin on its own at 0.25 µM did not show significant polymerization, i.e., increase in fluorescence. Therefore, we assume that the increase in fluorescence observed after mixing it with globular c-α-actins in G-buffer was solely due to the polymerization of the c-α-actins. Polymerization was induced by the addition of 2 mM MgCl₂ and 0.1 M KCl. The increase in pyrene-fluorescence was monitored using a Schimadzu RF5001PC or a Perkin-Elmer spectrofluorometer with excitation and emission wavelength settings of 365 nm and 385 nm, respectively [34 (link),35 (link)]. De-polymerization of F-c-actins induced by MICAL-1 was determined similarly with 5 µM F-c-actin variant supplemented with pyrene-labeled skeletal muscle actin by measuring the decrease in fluorescence after addition of NADPH [24 (link),35 (link)].

The HDL (2 mg/mL) was mixed with fructose (final 250 mM) in potassium phosphate/0.02% sodium azide buffer (pH 7.4) in the presence and absence of rHDL following the previously described method [17 (link)]. The mixture was incubated for 4 h at 37 °C in 5% atmospheric CO₂. The degree of advanced glycation reactions was evaluated by monitoring the fluorescent intensity using a spectrofluorometer (Perkin-Elmer, Norwalk, CT, USA) at the excitation wavelength of 370 nm and emission wavelength of 440 nm following the previously described method [36 (link)].

Malondialdehyde (MDA) was measured with the thiobarbituric acid colorimetric assay in the tissues.27 Briefly, 1 mL 10% (w/v) trichloroacetic acid was added to 450 μL of tissue lysate. After centrifugation, 1.3 mL 0.5% (w/v) thiobarbituric acid was added and the mixture was heated at 80°C for 20 minutes. After cooling, MDA formation was recorded (absorbance 530 nm and absorbance 550 nm) in a Perkin Elmer spectrofluorometer and the results were presented as ng MDA/mL.

The chymotrypsin-like activity of the proteasome in intact sperm was analyzed using the fluorescent substrate (Suc-LLVY-MCA). 2 × 10⁵ intact sperms were incubated in TYH medium without BSA, and 50 μmol/L fluorescent substrate for 30 min at 37 °C. The fluorescence was measured with excitation at 380 nm and emission at 460 nm using a spectrofluorometer (Perkin Elmer).

The chymotrypsin-like activity of proteasomes in the testes and epididymides was determined using a fluorescence substrate, Suc-LLVY-MCA (Peptide Institute, Osaka, Japan). Testicular and epididymal tissue extracts were incubated with 50 μM fluorescence substrate for 30 min at 37 °C. Fluorescence was measured via excitation at 380 nm and emission at 460 nm using a spectrofluorometer (PerkinElmer Inc., Waltham, MA, USA). The results were estimated as fluorescence intensity per protein amount and reaction time.

The fluorescent marker, fluorescein diacetate dye (FDA) (Sigma Chem., USA), was incorporated into ASH-BPNC. Then, ASH-BPNC loaded with FDA (ASH-BPNC-FDA) was orally administered (equivalent to 100 mg/ kg of ASH) to normal rats (n = 6). After 12 h, animals were sacrificed, and 100 mg of vital organs (brain, liver, kidney, and lungs) were excised and homogenized in ice-cold PBS. The accumulation of ASH-BPNC-FDA was detected in the resulting tissue homogenates using spectrofluorometer (PerkinElmer, Germany) at ʎexc:490 nm and ʎem:520 nm, respectively.
Furthermore, the accumulation of ASH-BPNC-FDA in tissues of vital organs were confirmed using in vivo fluorescent imaging, where sections of the remaining parts of the excised vital organs (brain, liver, kidney, and lungs) were preceded for Tissue-Tek Cryosat Microtome (Thermo Fisher, USA). Sections were examined under fluorescent microscope (IX81, Olympus, Tokyo, Japan) at magnification power × 200, and photomicrographs were captured.

Fluorescence emission was assessed for GO samples at concentration range of 25–200 µg mL⁻¹ using a LS‐50B Perkin Elmer Spectrofluorometer, at the excitation wavelength of 525 nm, with both excitation and emission slits set at 20.