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Microglia-Mediated Prevention of Traumatic Neurodegeneration
Published in Martin Berry, Ann Logan, CNS Injuries: Cellular Responses and Pharmacological Strategies, 2019
Solon Thanos, Rita Naskar, Peter Heiduschka
After induced microglial responses to injury34,35 phagocytosis-dependent labelling is a relatively new approach with several future perspectives. The fundamental advantage of function-dependent staining is that a number of questions about the role of microglia in neurodegeneration can be answered by studying their interaction with prelabelled, degenerating neurons in situ (Figure 7.1, A to H). Applied to RGC, such dyes result in function-dependent tracing of the nonneuronal cell scavengers which are the resident microglia. This convenient technique takes advantage of the neurons’ ability to internalise and transport material either from the terminals or from transected axons back to their cell bodies. Among the variety of dyes available for this purpose, the lipophilic carbocyanine group, originally used for membrane motility studies,36 turned out to be applicable in various experimental models including the retina (Figure 7.1). The use of dyes of different excitation/emittance wavelengths (Figure 7.1, I to K), enables double labelling such as in the developing and traumatised retina of rats.34,35 Dyes can be applied in the form of solid crystals anywhere within the nervous system and have the advantage of ensuring fast, vital labelling of the corresponding neurons (Figure 7.1, A to H). Long-term distribution of microglia can be examined both in the whole-mounted tissue (Figure 7.1) and in retinal sections processed for immunohistochemistry.36
Tumor Spheroids as a Model in Studies of Drug Effects
Published in Rolf Bjerkvig, Spheroid Culture in Cancer Research, 2017
Jörgen Carlsson, Thore Nederman
The first is to separate the spheroids by trypsination into a single-cell suspension and make a clonogenic survival assay on all the cells. This gives a mean survival at a certain drug exposure, and different subpopulations can be studied by varying the dose. Fluorescence-activated cell sorting based on gradients of intracellular probes for viable cells has been used to distinguish peripheral from central cells.32–34 Olive and Durand35 have recently described a carbocyanine derivative (DiOC7-3) that seems even better than the previously used Hoescht 33342 for characterizing the original positions of cells in spheroids after enzymatic treatment.
Optical Imaging Probes
Published in Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman, Molecular Imaging in Oncology, 2008
The major impact on fluorescence imaging in live animals and beyond was made as a result of introduction of carbocyanine dye derivatives that could be covalently linked to amino groups of proteins and other macro-molecules. Initially, isothiocyanate and iodoacetamide derivatives of several Cy dyes were shown to form covalent bonds with amino- and sulfhydryl groups of proteins and other macromolecules (58). These derivatives had insufficient hydrophilicity and were replaced by improved hydroxysuccinimide esters carboxylated dyes (59) and eventually were significantly improved by adding sulfate group substitutions in aromatic rings to increase solubility (60). The family of sulfated dyes included Cy5, which could be excited with the 633-nm HeNe laser and laser diodes emitting near 650 nm that are excellent excitation sources for Cy5 and related Cy5.5 dyes. The major application for some of these “early” Cy dye derivatives was found in labeling of oligonucleotides for genomic hybridization assays that allowed multiplexing because Cy3 and Cy5 dye fluorescence could be easily spectrally resolved.
The effects of neuronal cell differentiation on TRPM7, TRPM8 and TRPV1 channels in the model of Parkinson’s disease
Published in Neurological Research, 2022
For the measurement of mitochondrial membrane depolarization levels, the 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1 dye, Santa Cruz Biotechnology, USA) was used. The neuronal cells were incubated with 1 µM JC-1 dye for 30 min at 37°C as previously described [25]. This carbocyanine dye accumulates in the mitochondria via membrane potential-dependent. When membrane potential of mitochondria increases, the probe color changes from green to red. This fluorescence intensity ratio is used for indicating mitochondrial membrane depolarization. After incubation, the dye was removed by washing the cells with 1X PBS. The green JC-1 signal was measured at excitation (485 nm) and emission (535 nm) wavelengths. For the red signal, excitation (540 nm) and emission (590 nm) wavelengths were used. Fluorescence changings were analyzed using a fluorescence microplate reader (Tecan Infinite M200 Pro, Austria). Data were presented as emission values’ ratio (590/535) and calculated as fold increase experimental/control.
Preservation of small extracellular vesicles for functional analysis and therapeutic applications: a comparative evaluation of storage conditions
Published in Drug Delivery, 2021
Jun-Yong Wu, Yong-Jiang Li, Xiong-Bin Hu, Si Huang, Da-Xiong Xiang
To study the effect of storage conditions on the biodistribution of sEVs, healthy male BALB/c nude mice were employed as animal models. Isolated sEVs were labeled by carbocyanine dye DiR (Yeasen Biotechnology, China) for in vivo visualization (Li et al., 2020). Briefly, 10 μg of DiR was added to isolated sEVs fresh or after storage. After 20 min of incubation, unbounded DiR dye was removed by ultracentrifugation. DiR-labeled sEVs was resuspended in PBS. 100 μL of DiR-labeled sEVs were administrated to mice through tail vein injection and fluorescence was obtained using an AniView100 multimodal imaging system (Biolight Biotechnology Co., Ltd., China) at different time points. Ex vivo biodistribution was inspected after in vivo biodistribution monitoring. The animal study was carried out using the Institutional Animal Care and Use Committee (IACUC)-approved procedures. Animals were purchased from SJA Laboratory Animal Co., LTD (Hunan, China) and housed according to the regulations of the IACUC.
Targeting the gut microbial metabolic pathway with small molecules decreases uremic toxin production
Published in Gut Microbes, 2020
Yingyi Wang, Jianping Li, Chenkai Chen, Jingbo Lu, Jingao Yu, Xuejun Xu, Yin Peng, Sen Zhang, Shu Jiang, Jianming Guo, Jinao Duan
The transport of exogenous tryptophan into bacteria requires the synergism of proton-motive force, which is generated by electron transport chains embedded in the bacterial membrane.38 Thus, we were curious to understand whether ISO inhibits tryptophan transport by decreasing the proton-motive force. We used fluorescence microscopy and a membrane potential probe, carbocyanine dye DiOC2 (3; 3,3ʹ-Diethyloxacarbocyanine Iodide) to analyze the cell membrane potential. At low concentrations, DiOC2(3) emits a green fluorescence in bacterial cells, but it becomes concentrated in cells with a normal membrane potential, causing the dye to self-associate and the fluorescence emission to shift to red. Freshly incubated E. coli cells exhibit medium-red staining by DiOC2(3), indicating a normal state of membrane potential (Figure 3(a,b)). After treatment with ISO for 1 h, bacteria showed weaker red fluorescence intensity, indicating decreased membrane potential. According to the ImageJ analysis, the proportion of red fluorescence intensity decreased from 20.08% to 6.69%. Similar results were obtained when bacteria were treated with the membrane potential dissipating agent carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for 5 min.