Detecting and Destroying Cancer Cells in More Than One Way with Noble Metals and Different Confinement Properties on the Nanoscale *
Valerio Voliani in Nanomaterials and Neoplasms, 2021
Gold nanoparticles are also highly useful probes for microscopic imaging-based applications. For example, the resonant optical scattering intensity from a single 80 nm gold nanoparticle [15] is equivalent to the emission intensity of 500,000 of the most efficient Alexa Fluor dyes or 2000 of the most efficient Qdot 800 quantum dots [16]. While gold nanoparticles have been used for decades as labels in immunohistochemical and electron microscopic analysis of tissue sections [17], Sokolov et al. first demonstrated in 2003 that the resonant scattering from gold nanoparticles could be used to image subcellular cancer biomarkers (EGFR) in vitro using confocal reflectance microscopy of immunolabeled gold nanoparticles (ca. 12 nm core diameter) for potential cancer diagnostics, staging, and treatment monitoring. Our group [18, 19] has shown that simpler, less expensive dark-field optics can also be used to obtain high-contrast scattering images from immunolabeled gold nanoparticles in vitro, in this case in true-color, to achieve the identification and selective (photothermal) labeling of malignant cells (Fig. 1.2a). Currently, the method is in wide use for both the imaging and spectroscopy of nanoparticles and their labeling of living cells and tissues [20, 21].
Routine and Special Techniques in Toxicologic Pathology
Pritam S. Sahota, James A. Popp, Jerry F. Hardisty, Chirukandath Gopinath, Page R. Bouchard in Toxicologic Pathology, 2018
Traditional fluorochromes that are commonly used include fluorescein isothiocyanate (FITC, green color), 4′,6-diamidino-2-phenylindole (DAPI, blue color, binds to DNA in nuclei), and Texas Red (red color). Today, there are hundreds of fluorochromes available with various excitation peak and emission wavelengths. Desirable features of fluorochromes include a large extinction coefficient (likelihood of absorption of the excitation light), high quantum yield (ratio of light emitted to light absorbed, higher = brighter fluorescence), narrow emission spectrum (to minimize overlapping emissions when using multiple fluorochromes in a specimen), and good resistance to photobleaching (the irreversible decomposition of the fluorochrome by light excitation). Newer fluorochromes that possess more of these desirable features include cyanine dyes, Alexa Fluor dyes, DyLight fluorescent dyes, and Oyster fluorescent dyes.
Detection of Lysosomal Membrane Permeabilization
Bruno Gasnier, Michael X. Zhu in Ion and Molecule Transport in Lysosomes, 2020
Due to the reliance on the fluorescence of the Alexa Fluor label in this method, one has to consider if any other fluorescence arising from autofluorescence of the cells, treatment with a fluorescent small molecule or overexpression of a fluorescently tagged protein might interfere with the detection of the Alexa Fluor. Luckily, dextran coupled to many different Alexa Fluor labels is available, which makes it possible to solve this issue.
In vitro evaluation of archaeosome vehicles for transdermal vaccine delivery
Published in Journal of Liposome Research, 2018
Yimei Jia, Michael J. McCluskie, Dongling Zhang, Robert Monette, Umar Iqbal, Maria Moreno, Janelle Sauvageau, Dean Williams, Lise Deschatelets, Zygmunt J. Jakubek, Lakshmi Krishnan
Skin samples were frozen at −80 °C and cryosectioned of 20 µm thickness without fixation. Skin sections were mounted on glass slide with cover slip. Fluorescence microscopy was performed using an Olympus IX81 Microscope (Olympus Canada Inc., Toronto, ON, Canada). The X-Cite 120Q fluorescence light source (Excelitas Technologies, Waltham, MA, USA) link to the microscope through an optical fiber was used to excite all fluorophores used in this study. All emission/excitation cubes were from Semrock Inc (Rochester, NY). Fluorescence of Alexa Fluor 647 was detected using an excitation filter 628/40 and the emission at 692/40 (cat#: Cy5–4040 C). The excitation of rhodamine-DHPE was through a 543/22 filter and collected at 593/40 (Cat#: TRITC-B). For FITC and Dapi cube catalog no was FITC-2024 F-000 and DAPI-1160B-000, respectively. Each skin sample was imaged in triplicate using separate formulation preparations. The fluorescence intensity in different regions of skin was semi-quantified by line profile analysis using CellSens software (Olympus Corporation). Two areas to be quantified in each image were manually chosen, within which six equidistant lines were drawn to provide a total of 36 line profiles per formulation, and used to calculate the mean fluorescent intensity and diffusion depth.
Circular RNA circ_0010235 sponges miR-338-3p to play oncogenic role in proliferation, migration and invasion of non-small-cell lung cancer cells through modulating KIF2A
Published in Annals of Medicine, 2021
Yanan Zhu, Chunling Ma, Aiai Lv, Changwei Kou
Enhanced CCK-8 (Beyotime, Shanghai, China) and BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime, Shanghai, China) were utilized to measure NSCLC cell proliferation. Briefly, 3000 cells were transferred in 96-well plate, and five paralleled wells were set in each group. These adherent cells were further cultivated for 72 h, and 10 μL CCK-8 solution was added in each well at different time-points 0 h, 24 h, 48 h and 72 h. With 2 h-incubation of CCK-8, optical density (OD) at 450 nm was measured on a microplate reader. Cell proliferation curve was drawn according to OD values. For EdU assay, 5000 cells were transferred in 96-well plate in triplicate. After cell adherence, cells were dyed with 10 μM EdU reagent for 2 h and re-dyed with DAPI Staining Solution (Beyotime, Shanghai, China). Fluorescence detections of Alexa Fluor 488-labelled EdU and DAPI were performed on an inverted fluorescence microscope (Nikon Microsystems, Shanghai, China). EdU positive cell rate was calculated according to three random fields.
Icariin improves brain function decline in aging rats by enhancing neuronal autophagy through the AMPK/mTOR/ULK1 pathway
Published in Pharmaceutical Biology, 2021
Jie Zheng, Shanshan Hu, Jinxin Wang, Xulan Zhang, Ding Yuan, Changcheng Zhang, Chaoqi Liu, Ting Wang, Zhiyong Zhou
After melting paraffin at 60 °C for 4 h, the brain sections were deparaffinized and rehydrated conventionally. Sections were heated with citric acid buffer using microwave for 20 min, then incubated with 3% H2O2 for 10 min at room temperature (RT). After blocking with 5% BSA for 1 h at RT, the tissue sections were incubated with primary antibody (anti-LC3 and anti-NeuN) diluted in 1% BSA and 0.5% Triton X-100 overnight at 4 °C. Followed by rewarming for 30 min at RT, washing with PBS. Corresponding Alexa Fluor antibody with fluorescence were incubated for 1 h avoiding light at RT. After washing, the tissue sections were stained with DAPI at 25 °C for 10 min. Finally, adding anti-fluorescence quencher agent on the sections and sealing. Image acquisition under confocal microscope (Nikon, A1R+).
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