Notes

Omental hair loss transplant regarding neuroendocrinological problems.
Herein we describe the common aspects of indirect methods and propose a general step-by-step procedure for the determination of ΦΔ values. In addition, we identify the key experimental conditions that need to be controlled to obtain meaningful results.Spin trapping with cyclic nitrones coupled to electron paramagnetic resonance (EPR) enables the detection and characterization of oxygen-derived free radicals, such as superoxide and hydroxyl radicals, in living cells. Detection is usually performed on cell suspensions introduced in glass capillaries, gas-permeable tubing, or flat cells, even when cells normally require attachment for growth. However, radical production may be influenced by cell adhesion, while enzymatic or mechanical cell harvesting may damage the cells and alter their metabolic rates. Here, we describe the detection on adherent cells attached to microscope coverslip glasses. This method preserves cell integrity, ensures near physiological conditions for naturally adherent cells, and is relatively simple to set up. Up to 12 conditions can be screened in half a day using a single batch of culture cells.Electron paramagnetic resonance (EPR) spectroscopy is an established method for the measurement of free radicals. Solar radiation is essential for human life as it stimulates vitamin D synthesis and well-being. However, an excessive dose of solar radiation leads to the formation of free radicals. Here, we describe an EPR method for measuring the amount of radicals induced by UVA irradiation in excised skin. For the first time, a wavelength stable UVA LED (365 nm) was used. The method allows the quantitative determination of radicals in skin before, during, and after UVA irradiation. A dose-dependent radical production could be demonstrated, independent of the yielded power.Reactive oxygen species (ROS) production within biofilms is studied with a simple and easy setup based on fluorescence microscopy. check details Herein, a biofilm is exposed to different ROS inducers a bactericidal antibiotic (ciprofloxacin) and a BODIPY-based photosensitizer (I2B-OAc). Real-time ROS induction in the core of the biofilms is monitored utilizing two fluorescent reporters-AMDA and H2DCFDA-the first one with selectivity toward singlet oxygen (1O2) and the latest for other ROS (O2•-, H2O2, and OH•-). A point-by-point methodology is reported, starting with the sample preparation all the way through the microscope setup and, finally, processing of the images.Different experimental conditions can be used to detect the presence of reactive oxygen species (ROS) in the photodynamic inactivation of microorganisms. Here, we describe the effect of the media and the addition of ROS scavengers to obtain insight about the oxidative processes that take place during the photokilling of bacteria. In addition, 9,10-dimethylanthracene was used to sense the generation of singlet molecular oxygen, O2(1Δg), in microbial cells. Thus, the contribution of type I or type II pathways in the photocytotoxicity action can be rapidly detected and compared between different photosensitizers.Reactive oxygen species (ROS) are continuously produced in semen and are essential for important spermatozoa functions that allow fertilization. However, an excessive amount of ROS is associated with poor sperm quality, which can compromise male fertility potential. This chemiluminescence assay is based on the production of light through the reaction between luminol and ROS. The emitted light is converted to an electrical signal by a luminometer, and the ROS levels in the sample are calculated as relative light units (RLU) per second per million spermatozoa per milliliter (RLU/s/million sperm/mL).Reactive oxygen species (ROS) could have a negative impact on sperm cellular function and viability. This chapter describes a protocol for oxidative stress evaluation using dichlorofluorescein (DCF) which can specifically reveal intracellular reactive oxygen species. The protocol described here has been used in human and teleost species sperm samples. The method can be used with two approaches (1) flow cytometry, for quantification of DCF+ cells, or (2) confocal microscopy, for the localization of ROS within the cells.The budding yeast Saccharomyces cerevisiae is a facultative organism that is able to utilize both anaerobic and aerobic metabolism, depending on the composition of carbon source in the growth medium. When glucose is abundant, yeast catabolizes it to ethanol and other by-products by anaerobic fermentation through the glycolysis pathway. Following glucose exhaustion, cells switch to oxygenic respiration (a.k.a. "diauxic shift"), which allows catabolizing ethanol and the other carbon compounds via the TCA cycle and oxidative phosphorylation in the mitochondria. The diauxic shift is accompanied by elevated reactive oxygen species (ROS) levels and is characterized by activation of ROS defense mechanisms. Traditional measurement of the diauxic shift is done through measuring optical density of cultures grown in a batch at intermediate time points and generating a typical growth curve or by estimating the reduction of glucose and accumulation of ethanol in growth media over time. In this manuscript, we describe a method for determining changes in ROS levels upon yeast growth, using carboxy-H(2)-dichloro-dihydrofluorescein diacetate (carboxy-H(2)-DCFDA). H2-DCFDA is a widely used fluorescent dye for measuring intracellular ROS levels. H2-DCFDA enables a direct measurement of ROS in yeast cells at intermediate time points. The outcome of H2-DCFDA fluorescent readout measurements correlates with the growth curve information, hence providing a clear understanding of the diauxic shift.Autophagy constitutes an essential process triggered by oxidative stress that enables cells to recycle damaged biomolecules and organelles, which is eventually traced by immunodetection with anti-ATG8. In parallel with autophagy induction, carbon metabolism in Chlamydomonas reinhardtii under abiotic stress is diverged toward lipid biosynthesis and lipid droplet accumulation, which can be analyzed by a simple thin-layer chromatography and in vivo staining with the fluorescent probe BODIPY 493/503. We show the responses in Chlamydomonas cells exposed to mercury or cadmium (0-50 μM doses), as examples of oxidative stress-mediated changes in autophagy and lipid metabolism, monitored with the procedures described in this report.
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