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Noninvasive Sensing of Serum sRAGE and Glycated Hemoglobin by Skin UV-Induced Fluorescence
Published in Andrey V. Dunaev, Valery V. Tuchin, Biomedical Photonics for Diabetes Research, 2023
Vladimir V. Salmin, Tatyana E. Taranushenko, Natalya G. Kiseleva, Alla B. Salmina
We developed the original compact spectrofluorometer with UV LED excitation for the assessment of skin autofluorescence. The device (Figure 7.1) includes the following elements: Information about the spectra can be obtained using the spectrometer STS-VIS USA (1). Radiation from the object under study enters the spectrometer through a condenser lens (2). To excite fluorescence, frontal illumination of UV LEDs with different wavelengths, crossing beams at an acute angle (3–5), is used. A white LED (6) is used to record the reflection spectra. For synchronous with the start of the spectrometer on and off the LEDs, a hardware-software device for controlling LEDs is used (7). The measurement signal is triggered by a command from the computer. For the convenience of storing information (measurement data, software), a flash memory module is used (8). A USB hub (9) is used to connect USB devices. Data transfer to a computer and power supply of the device is carried out via a USB cable (10). Fixation of the device on the skin area is carried out using a lens hood (11).
Pre-Clinical In-Vivo and In-Vitro Methods For Evaluation of Anti-Alzheimer’s Drugs
Published in Atanu Bhattacharjee, Akula Ramakrishna, Magisetty Obulesu, Phytomedicine and Alzheimer’s Disease, 2020
Shilpa A. Deshpande, Niraj S. Vyawahare
Aliquots of 0.05 ml 0.4 M HCl and 0.1 ml of EDTA/sodium acetate buffer (pH 6.9) are added to 0.2 ml of the aqueous phase, followed by 0.1 ml of 0.1 M iodine solution (in ethanol) for oxidation. The reaction is stopped after 2 min by addition of 0.1 ml Na2SO3 solution, and 0.1 ml acetic acid is added after a further 1.5 min. The solution is then heated to 100°C for 6 min. Excitation and emission spectra are recorded from the spectrofluorometer when the sample reaches room temperature. The readings are taken at 330 (excitation) and 375 nm (emission) for dopamine and at 395 and 485 nm for noradrenaline.
Collagenolysis
Published in Robert A. Greenwald, CRC Handbook of Methods for Oxygen Radical Research, 2018
James Paul Borel, Jean Claude Monboisse
After incubation of fibrils in the presence of a system generating oxy free radicals, the suspension is centrifuged at 35,000 × g at 4°C for 30 min. The supernatant is separated and the pellet resuspended in the same 0.1 M tris buffer, pH 7.4 and centrifuged again. The two supernatants are combined, the volume measured, and an aliquot of 2 mℓ hydrolyzed in 6 M HCl for 24 hr at 115°C. Proline and hydroxyproline are separated from mineral ions by ion exchange chromatography on a Dowex 50 W × 2 column. The amounts of these amino acids are evaluated by a quantitative method sensitive at the picomole level.13 Briefly, the amino acids are combined with the fluorophor 7-chloro-4-nitrobenzo-2-oxy-1,3-diazole (NBD-C1) which gives stable fluorescent derivatives only with cyclic amino acids such as hydroxyproline. The fluorescent derivatives are separated by thin layer chromatography and quantitated in a Farrand spectrofluorometer equipped with a thin layer chromatography scanning device. Control samples incubated without reagents, or in the presence of SOD (Sigma, 5 units per milliliter) or catalase (Worthington #1872, 83 units per milliliter) must always be processed in the same conditions.
Comparative evaluation of the effects of bisphenol derivatives on oxidative stress parameters in HepG2 cells
Published in Drug and Chemical Toxicology, 2023
Busra Ozyurt, Gizem Ozkemahli, Anil Yirun, Aylin Balci Ozyurt, Merve Bacanli, Nursen Basaran, Belma Kocer-Gumusel, Pinar Erkekoglu
The method is based on the measurement of the bright green fluorescence (λex = 490/λem = 520 nm) resulting from the conversion of non-fluorescent 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) to green fluorescent 2′,7′-dichlorofluorescein (CM-DCF) by living cells. HepG2 cells were seeded in black/clear bottom 96-well plate at a density of 104 cells to per well in 90 µl. After the cells were seeded, they incubated for 24 h at 37 °C in an incubator; each test substance was added to the respective well at 10-fold concentration of their IC30 doses in 10 µl. Same amount of cell culture medium was added for the control group. To determine intracellular ROS production, cell plate was incubated in in the incubator with 5% CO2 for 1 h. Master reaction mix was added to each well and plate was incubated at 37 °C in the incubator with 5% CO2 for 45 min in dark. The fluorescence was measured by a spectrofluorometer SpectraMax M2 (Molecular Devices, Sunnyvale, CA). The amount of intracellular ROS produced by the control cells was assumed as 100%, and the amount of ROS produced by the other cells was calculated as percentage of the control.
Incorporation of electroendocytosis and nanosecond pulsed electric field in electrochemotherapy of breast cancer cells
Published in Electromagnetic Biology and Medicine, 2022
The control and the samples exposed to low-intensity electric pulses in the presence of BSA-FTIC were washed and centrifuged (210 g, 10 min, room temperature) twice in PBS. To release the cell lysate, the pellet was resuspended in 1 ml Lysis buffer (5x lysis reagent for cell culture, Promega, Charbonnieres, France) diluted to 1/100 with water and vortexed. A spectrofluorometer (SFM 25, Kontron Instruments, Herts, England) was used to measure the incorporated fluorescence, which represents the total fluorescence released by the ruptured cells. For fluorescence measurements, the excitation and emission wavelengths for BSA-FITC were 525 nm and 520 nm, respectively. The fluorescence measurements were normalized into number of molecules incorporated per cell using a fluorescence standard curve and the number of cells per sample. The uptake of BSA-FTIC by cells exposed to low intensity monopolar electric pulses was computed in comparison to the average uptake of control cells (considered to be 100%) exposed to the fluorescent probe only for the same duration, in the absence of the electric pulses. The dependence of receptor-mediated endocytosis on the field intensities (5–20 V/cm) of the monopolar electric pulses was monitored (Antov et al. 2004; 2005).
Hemoglobin–albumin cluster: physiological responses after exchange transfusion into rats and blood circulation persistence in dogs
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Hitomi Iwasaki, Kyoko Yokomaku, Moeka Kureishi, Keisuke Igarashi, Ryo Hashimoto, Mitsutomo Kohno, Masayuki Iwazaki, Risa Haruki, Motofusa Akiyama, Kenichi Asai, Yuka Nakamura, Ryosuke Funaki, Yoshitsugu Morita, Teruyuki Komatsu
After HSA was purchased from the Japan Blood Products Organization (Tokyo, Japan), it was purified using gel filtration chromatography (GPC) to remove the stabilizing agents. For exchange transfusion, a phosphate buffered saline (PBS, pH 7.4) solution of HSA was used. The Hb–HSA3 cluster comprising a bovine Hb core and an average of three HSAs at the exterior was synthesized according to our procedures reported earlier [21]. The Hb unit concentration of the Hb–HSA3 formulation is 5 g/dL in PBS (pH 7.4). At 37 °C, the P50 and Hill coefficient of Hb–HSA3 are 9 Torr and 1.4, respectively. A fluorescence labeling agent, Cyanine5.5 (Cy5.5) NHS ester, was purchased from Lumiprobe Corp (Maryland, USA). All other chemical reagents were purchased from commercial sources as special grades and were used without further purification. Deionized water (18.2 MΩcm) was prepared using water purification systems (Elix UV and Milli Q Reference; Millipore Corp.). The UV-visible absorption spectra were recorded using a UV–visible spectrophotometer (8543; Agilent Technologies Inc.) attached with a Peltier temperature controller (89090A; Agilent Technologies Inc.). Fluorescence emission spectra were measured using a spectrofluorometer (FP-6500; Jasco Corp.). The molecular size was determined by dynamic light scattering (DLS) measurement using a particle size analyzer (ELSZ-2000; Otsuka Electronics Co., Ltd.).