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A new-infrared probe targeting mitochondria via regulation of molecular hydrophobicity
摘要： Herein, we developed a near-infrared (NIR) fluorescent probe for mitochondrial staining based on the NIR fluorochrome, silicon-rhodamine. The hydrophobicity of the fluorescent core was systematically modified by conjugation with 10 different commercial amines. The resulting fluorescent compounds exhibited similar photophysical properties but diverse hydrophobicity. We identified the optimal level of hydrophobicity associated with high mitochondrial targeting efficiency. In particular, the SiR-Mito 8 probe provided excellent mitochondrial staining and successfully differentiated the live Hep3B cancer cells from normal L02 cells in vitro.
关键词： Mitochondria,silicon rhodamine,fluorescent probes,near-IR
更新于2025-09-23 15:19:57
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Measurement of ROS in Caenorhabditis elegans Using a Reduced Form of Fluorescein
摘要： Oxidative stress is implicated in the pathogenesis of various neurodegenerative diseases, including Alzheimer’s disease. Oxidative stress is a result of a disruption of the equilibrium between antioxidants and oxidants, in favor of oxidants. Since mitochondria are major sites of production and reduction of reactive oxygen species (ROS), measurement of ROS levels can help us determine if mitochondrial functional integrity has been compromised. In this protocol, we describe a method to measure the level of ROS in the nematode Caenorhabditis elegans, using chloromethyl-2,7’-dichlorodihydrofluorescein diacetate (CM-H2DCFDA).
关键词： CM-H2DCFDA,C. elegans,Mitochondria,Oxidative stress,DCF,ROS
更新于2025-09-19 17:15:36
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Imaging of anti-inflammatory effects of HNO <i>via</i> a near-infrared fluorescent probe in cells and in rat gouty arthritis model
摘要： Nitroxyl (HNO) plays a crucial role in anti-inflammatory effects via the inhibition of inflammatory pathways, but the details of the endogenous generation of HNO still remain challenging owing to the complex biosynthetic pathways, in which the interaction between H2S and NO simultaneously generates HNO and polysulfides (H2Sn) in mitochondria. Moreover, nearly all the available fluorescent probes for HNO are utilized for imaging HNO in cells and tissues, instead of the in situ real-time detection of the simultaneous formation of HNO and H2Sn in mitochondria and animals. Here, we have developed a mitochondria-targeting near-infrared fluorescent probe, namely, Mito-JN, to detect the generation of HNO in cells and a rat model. The probe consists of three moieties: Aza-BODIPY as a fluorescent signal transducer, a triphenylphosphonium cation as a mitochondria-targeting agent, and a diphenylphosphinobenzoyl group as an HNO-responsive unit. The response mechanism is based on an aza-ylide intramolecular ester aminolysis reaction with fluorescence emissions on. Mito-JN displays high selectivity and sensitivity for HNO over various other biologically relevant species. Mito-JN was successfully used for the detection of the endogenous generation of HNO, which is derived from the crosstalk between H2S and NO in living cells. The additional generation of H2Sn was also confirmed using our previous probe Cy-Mito. The anti-inflammatory effect of HNO was examined in a cell model of LPS-induced inflammation and a rat model of gouty arthritis. The results imply that our probe is a good candidate for the assessment of the protective effects of HNO in inflammatory processes.
关键词： H2S,fluorescent probe,HNO,polysulfides,gouty arthritis,anti-inflammatory,near-infrared,NO,mitochondria-targeting
更新于2025-09-19 17:15:36
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A highly selective and sensitive fluorescent probe for simultaneously distinguishing and sequentially detecting H <sub/>2</sub> S and various thiol species in solution and in live cells
摘要： A novel dual-channel fluorescent probe (NCR) based on differences in reactivity among H2S, Cys/Hcy, and GSH was rationally designed for simultaneously distinguishing and sequentially sensing H2S, Cys/Hcy, and GSH using two emission channels, which also demonstrated that NCR can be used for targeting mitochondria in mammalian cells.
关键词： live cells,thiol species,fluorescent probe,mitochondria-targeted,H2S
更新于2025-09-19 17:15:36
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Distribution and Activity of Mitochondrial Proteins in Vascular and Avascular Retinas: Implications for Retinal Metabolism
摘要： PURPOSE. Understanding the energetics of retinal neurons and glia is crucial for developing therapies for diseases that feature deficits in nutrient or oxygen availability. Herein, we performed a detailed characterization of the distribution and activity of mitochondrial proteins in the vascularized retinas of rat and marmoset, and the avascular retinas of rabbit and guinea pig. Further, we delineated expression of ubiquitous mitochondrial creatine kinase (uMtCK). METHODS. Expression of eight mitochondrial proteins was investigated using Western blotting, single- and double-labeling immunohistochemistry. Activities of cytochrome c oxidase, succinate dehydrogenase, and isocitrate dehydrogenase were determined by enzyme histochemistry using unfixed tissue sections. RESULTS. In vascularized retinas, immunoreactivities were characterized by strong, punctate labeling in the plexiform layers, photoreceptor inner segments, somas of various cell types, notably retinal ganglion cells (RGCs), and the basolateral surface of the retinal pigment epithelium. In avascular retinas, immunoreactivities featured intense labeling of inner segments, together with weak, but unambiguous, staining of both plexiform layers. RGCs were relatively enriched. In Müller cells of avascular retinas, mitochondria were restricted to scleral-end processes. For each species, enzyme activity assays yielded similar results to the protein distributions. Labeling for uMtCK in vascular and avascular retinas was fundamentally similar, being restricted to neuronal populations, most notably inner segments and RGCs. Of all of the mitochondrial proteins, uMtCK displayed the strongest labeling in avascular retinas. uMtCK was not detectable in Müller cells in any species. CONCLUSIONS. The current findings advance our understanding of the metabolic similarities and differences between vascular and avascular retinas.
关键词： mitochondrial creatine kinase,mitochondria,vascular,avascular,aerobic metabolism,retina
更新于2025-09-19 17:15:36
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Mitochondrial Respiration in Outer Retina Contributes to Light-Evoked Increase in Hydration In Vivo
摘要： PURPOSE. To test the hypothesis that mitochondrial respiration contributes to local changes in hydration involved in phototransduction-driven expansion of outer retina, as measured by structural responses on optical coherence tomography (OCT) and diffusion magnetic resonance imaging (MRI). METHODS. Oxygen consumption rate and mitochondrial reserve capacity of freshly isolated C57BL/6 and 129S6/SvEvTac mouse retina were measured using a Seahorse Extracellular Flux Analyzer. Light-stimulated outer retina layer water content was determined by proton density MRI, structure and thickness by ultrahigh-resolution OCT, and water mobility by diffusion MRI. RESULTS. Compared with C57BL/6 mice, 129S6/SvEvTac retina demonstrated a less robust mitochondrial respiratory basal level, with a higher reserve capacity and lower oxygen consumption in the light, suggesting a relatively lower production of water. C57BL/6 mice showed a light-triggered surge in water content of outer retina in vivo as well as an increase in thickness, and water mobility. In contrast, light did not evoke augmented hydration in this region or an increase in hyporeflective bands or water mobility in the 129S6/SvEvTac outer retina. Nonetheless, we observed a significant but small increase in outer retinal thickness. CONCLUSIONS. These studies suggest that respiratory-controlled hydration in healthy retina is linked with a localized light-evoked expansion of the posterior retina in vivo and may serve as a useful biomarker of the function of photoreceptor/retinal pigment epithelium complex.
关键词： mitochondria,water content,functional imaging,genetics,photoreceptor
更新于2025-09-19 17:15:36
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Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells
摘要： T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.
关键词： lysosome,Flow cytometry,organelle-specific dyes,mitochondria,multicolor,T cell
更新于2025-09-19 17:15:36
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Differential Responses of Myoblasts and Myotubes to Photobiomodulation are associated with Mitochondrial Number
摘要： Objective: Photobiomodulation (PBM) is the application of light to promote tissue healing. Current indications suggest PBM induces its beneficial effects in vivo through upregulation of mitochondrial activity. However, how mitochondrial content influences such PBM responses has yet to be evaluated. Hence, the current study assessed the biological response of cells to PBM with varying mitochondrial contents. Methods: DNA was isolated from myoblasts and myotubes (differentiated myoblasts) and mitochondrial DNA (mtDNA) was amplified and quantified using a microplate assay. Cells were seeded in 96-wellplates, incubated overnight and subsequently irradiated using a LED array (400nm, 450nm, 525nm, 660nm, 740nm, 810nm,830nm and white light, 24mW/cm2, 30-240s, 0.72-5.76J/cm2). The effects of PBM on markers of mitochondrial activity including reactive-oxygen-species (ROS) and real-time mitochondrial respiration (Seahorse XFe96) assays were assessed 8 h post-irradiation. Datasets were analysed using general linear model followed by one-way ANOVA (and post hoc-Tukey tests); p=0.05). Results: Myotubes exhibited mtDNA levels 86% greater than myoblasts (p<0.001). Irradiation of myotubes at 400nm, 450nm or 810nm induced 53%, 29% and 47% increases (relative to non-irradiated-control) in maximal respiratory rates respectively (p<0.001). Conversely, irradiation of myoblasts at 400nm or 450nm had no significant effect on maximal respiratory rates. Conclusion: This study suggests that mitochondrial content may influence cellular responses to PBM and as such explain the variability of PBM responses seen in the literature.
关键词： mitochondria,Low level light therapy,LLLT,Photobiomodulation,Myogenesis
更新于2025-09-19 17:15:36
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The Protozoan, <i>Paramecium primaurelia</i> , as a Non-sentient Model to Test Laser Light Irradiation: The Effects of an 808nm Infrared Laser Diode on Cellular Respiration
摘要： Photobiomodulation (PBM) has been used in clinical practice for more than 40 years. Unfortunately, conflicting literature has led to the labelling of PBM as a complementary or alternative medicine approach. However, past and ongoing clinical and research studies by reputable investigators have re-established the merits of PBM as a genuine medical therapy, and the technique has, in the last decade, seen an exponential increase in the numbers of clinical instruments available, and their applications. This resurgence has led to a clear need for appropriate experimental models to test the burgeoning laser technology being developed for medical applications. In this context, an ethical model that employs the protozoan, Paramecium primaurelia, is proposed. We studied the possibility of using the measure of oxygen consumption to test PBM by irradiation with an infrared or near-infrared laser. The results show that an 808nm infrared laser diode (1W; 64J/cm2) affects cellular respiration in P. primaurelia, inducing, in the irradiated cells, a significantly (p < 0.05) increased oxygen consumption of about 40%. Our findings indicate that Paramecium can be an excellent tool in biological assays involving infrared and near-infrared PBM, as it combines the advantages of in vivo results with the practicality of in vitro testing. This test represents a fast, inexpensive and straightforward assay, which offers an alternative to both traditional in vivo testing and more-expensive mammalian cellular cultures.
关键词： laser irradiation,mitochondria,low level laser therapy,high level laser therapy,photobiostimulation,laser phototherapy
更新于2025-09-16 10:30:52
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The 808?nm and 980?nm infrared laser irradiation affects spore germination and stored calcium homeostasis: A comparative study using delivery hand-pieces with standard (Gaussian) or flat-top profile
摘要： Photobiomodulation relies on the transfer of energy from incident photons to a cell photoacceptor. For many years the concept of photobiomodulation and its outcome has been based upon a belief that the sole receptor within the cell was the mitochondrion. Recently, it has become apparent that there are other photoacceptors operating in different regions of the electromagnetic spectrum. Alternative photoacceptors would appear to be water and mechanisms regulating calcium homeostasis, despite a direct effect of laser photonic energy on intracellular calcium concentration outwith mitochondrial activity or influence, have not been clearly demonstrated. Therefore, to increase the knowledge of intracellular?calcium and laser photon interaction, as well as to demonstrate differences in irradiation profiles with modern hand-pieces, we tested and compared the photobiomodulatory effect of 808 nm and 980 nm diode laser light by low- and higher-energy (60s, 100 mW/cm2, 100 mW/cm2, 500 mW/cm2, 1000 mW/cm2, 1500 mW/cm2, 2000 mW/cm2) irradiated with a “standard” (Gaussian fluence distribution) hand-piece or with a “flat-top” (uniform fluence) hand-piece. For this purpose, we used the eukaryote unicellular-model Dictyostelium discoideum. The 808 nm and 980 nm infrared laser light, at the energy tested directly affect the stored Ca2+ homeostasis, independent of the mitochondrial respiratory chain activities. From an organism perspective, the effect on Ca2+-dependent signal transduction as the regulator of spore germination in Dictyostelium, demonstrates how a cell can respond quickly to the correct laser photonic stimulus through a different cellular pathway than the known light-chromophore(mitochondria) interaction. Additionally, both hand-piece designs tested were able to photobiomodulate the D. discoideum cell; however, the hand-piece with a flat-top profile, through uniform fluence levels allows more effective and reproducible effects.
关键词： Mitochondria activities,Laser therapy,Photobiomodulation,Low-level laser therapy,Intracellular calcium concentration
更新于2025-09-16 10:30:52