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Cell Navigator® Lysosome Staining Kit *Green Fluorescence with 405 nm Excitation*

Image of U2OS cells stained with Cell Navigator® Lysosomal Staining Kit in a Costar black wall/clear bottom 96-well plate.
Image of U2OS cells stained with Cell Navigator® Lysosomal Staining Kit in a Costar black wall/clear bottom 96-well plate.
To determine whether genistein or PP2 alters the distribution of the internalized BmCPV particles, we used confocal microscopy to locate internalized BmCPV virions and lysosomes. Genistein and PP2 route BmCPV to the wrong destination- lysosomes. (a) Normal BmN cells (control) were incubated with A546-labeled BmCPV virions and a lysosomal staining agent for 3 h; (b) BmN cells were treated with PP2 (0.16 μM) for 30 min, followed by incubation with A546-labeled BmCPV virions and a lysosomal staining agent for 3 h; (c) BmN cells were treated with genistein (50 μg/mL) for 1 h, followed by incubation with A546-labeled BmCPV virions and a lysosomal staining agent for 3 h; (d) quantification of the spectral overlap of BmCPV particles with lysosomes. The spectral overlap is presented as the percent co-localization (n = 21 cells). Error bars indicate standard deviations. ***P < 0.001. Source: <strong>Clathrin-mediated endocytosis is a candidate entry sorting mechanism for Bombyx mori cypovirus </strong>by Chen et al., <em>Scientific Reports, </em>May 2018.
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Catalog Number22651
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Telephone1-408-733-1055
Fax1-408-733-1304
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H-phraseH303, H313, H333
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Intended useResearch Use Only (RUO)
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UNSPSC12352200

OverviewpdfSDSpdfProtocol


Our Cell Navigator® fluorescence imaging kits are a set of fluorescence imaging tools for labeling sub-cellular organelles such as membranes, lysosomes, mitochondria and nuclei etc. The selective labeling of live cell compartments provides a powerful method for studying cellular events in a spatial and temporal context. This particular kit is designed to label lysosomes of live cells in green fluorescence. The kit uses a proprietary lysotropic dye that selectively accumulates in lysosomes probably vial the lysosome pH gradient. The lysotropic indicator is a hydrophobic compound that easily permeates intact live cells, and trapped in lysosomes after it gets into cells. Its fluorescence is significantly enhanced upon entering lysosomes. This key feature significantly increases its selectivity for lysosomes. The labeling protocol is robust, requiring minimal hands-on time. It can be readily adapted for a wide variety of fluorescence platforms such as microplate assays, immunocytochemistry and flow cytometry. It is useful for a variety of studies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. The kit provides all the essential components with an optimized cell-labeling protocol. It is suitable for proliferating and non-proliferating cells, and can be used for both suspension and adherent cells. This kit is optimized for flow cytometers equiped with viloet laser excitation @ 405 nm and emission around 500 nm.

Platform


Fluorescence microscope

Excitation405 nm
Emission480 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Violet filter set

Components


Component A: LysoBrite™ VLG261 vial (100 µL, 500X DMSO stock solution)
Component B: Live Cell Staining Buffer1 bottle (50 mL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells
  2. Add LysoBrite™ VLG26 working solution
  3. Incubate at 37°C for 30 minutes to 2 hours
  4. Analyze the cells under fluorescence microscope at Ex/Em = 405/480 nm (Violet filter set)

Important notes
Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF WORKING SOLUTION

Add 20 µL of 500X LysoBrite™ VLG26 stock solution (Component A) to 10 mL of Live Cell Staining Buffer (Component B) and mix well to make LysoBrite™ VLG26 working solution. Protect from light. Note: 20 µL of 500X LysoBrite™ VLG26 stock solution (Component A) is enough for one 96-well plate. The optimal concentration of the fluorescent lysosome indicator varies depending on the specific application. The staining conditions may be modified according to the particular cell type and the permeability of the cells or tissues to the probe.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

For adherent cells:

  1. Grow cells either in a 96-well black wall/clear bottom plate (100 µL/well/96-well plate) or on coverslips inside a petri dish filled with the appropriate culture medium. When cells reach the desired confluence, add equal volume (such as 100 µL/well/96-well plate) of LysoBrite™ VLG26 working solution.

  2. Incubate the cells in a 37°C, 5% CO2 incubator for 30 minutes to 2 hours.

  3. Observe the cells using a fluorescence microscope with Violet filter set (Ex/Em = 405/480 nm). Note: It is recommended to increase either the labeling concentration or the incubation time to allow the dye to accumulate if the cells do not appear to be sufficiently stained.

For suspension cells:

  1. Centrifuge the cells at 1,000 rpm for 5 minutes to obtain a cell pellet and aspirate the supernatant.

  2. Resuspend the cell pellet gently in pre-warmed growth medium, and then add equal volume of LysoBrite™ VLG26 working solution.

  3. Incubate the cells in a 37°C, 5% CO2 incubator for 30 minutes to 2 hours.

  4. Observe the cells using a fluorescence microscope with Violet filter set (Ex/Em = 405/480 nm). Note: It is recommended to increase either the labeling concentration or the incubation time to allow the dye to accumulate if the cells do not appear to be sufficiently stained. Suspension cells may be attached to coverslips that have been treated with BD Cell-Tak® (BD Biosciences) and stained as adherent cells.

Citations


View all 19 citations: Citation Explorer
Photocrosslinked Bioreducible Polymeric Nanoparticles for Enhanced Systemic siRNA Delivery as Cancer Therapy
Authors: Karlsson, Johan and Tzeng, Stephany Y and Hemmati, Shayan and Luly, Kathryn M and Choi, Olivia and Rui, Yuan and Wilson, David R and Kozielski, Kristen L and Qui{\~n}ones-Hinojosa, Alfredo and Green, Jordan J
Journal: Advanced Functional Materials (2021): 2009768
Upconversion nanoparticles as intracellular pH messengers
Authors: Tsai, Evaline S and Joud, Fadwa and Wiesholler, Lisa M and Hirsch, Thomas and Hall, Elizabeth AH
Journal: Analytical and Bioanalytical Chemistry (2020): 1--15
Increasing Uptake of Silica Nanoparticles with Electroporation: From Cellular Characterization to Potential Applications
Authors: Phonesouk, Erick and Lechevallier, Séverine and Ferr, undefined and , Audrey and Rols, Marie-Pierre and Bezombes, Christine and Verelst, Marc and Golzio, Muriel
Journal: Materials (2019): 179
Understanding intracellular trafficking and anti-inflammatory effects of minocycline chitosan-nanoparticles in human gingival fibroblasts for periodontal disease treatment
Authors: Martin, Victor and Ribeiro, Isabel AC and Alves, Marta M and Gon{\c{c}}alves, L{\'\i}dia and Almeida, Ant{\'o}nio J and Grenho, Liliana and Fernandes, Maria H and Santos, Catarina F and Gomes, Pedro S and Bettencourt, Ana F
Journal: International journal of pharmaceutics (2019): 118821
Clathrin-mediated endocytosis is a candidate entry sorting mechanism for Bombyx mori cypovirus
Authors: Chen, Fei and Zhu, Liyuan and Zhang, Yiling and Kumar, Dhiraj and Cao, Guangli and Hu, Xiaolong and Liang, Zi and Kuang, Sulan and Xue, Renyu and Gong, Chengliang
Journal: Scientific reports (2018): 7268
Staphylococcus aureus from atopic dermatitis skin accumulates in the lysosomes of keratinocytes with induction of IL-1α secretion via TLR 9
Authors: Moriwaki, Masaya and Iwamoto, Kazumasa and Niitsu, Yoshie and Matsushima, Ayako and Yanase, Yuhki and Hisatsune, Junzo and Sugai, Motoyuki and Hide, Michihiro
Journal: Allergy (2018)
Silica-Based Nanoparticles as Bifunctional and Bimodal Imaging Contrast Agents
Authors: Lechevallier, S{\'e}verine and Mauricot, Robert and Gros-Dagnac, H{\'e}l{\`e}ne and Chevreux, Sylviane and Lemercier, Gilles and Phonesouk, Erick and Golzio, Muriel and Verelst, Marc
Journal: ChemPlusChem (2017): 770--777
Silica-based nanoparticles as bi-functional and bi-modal imaging contrast agents
Authors: Lechevallier, Séverine and Mauricot, Robert and Gros-Dagnac, Hélène and Chevreux, Sylviane and Lemercier, Gilles and Phonesouk, Erick and Golzio, Muriel and Verelst, Marc
Journal: ChemPlusChem (2017)
A Triple-Fluorophore Labeled Nucleic Acid pH Nanosensor to Investigate Non-Viral Gene Delivery
Authors: Wilson, David R and Routkevitch, Denis and Rui, Yuan and Mosenia, Arman and Wahlin, Karl J and Quinones-Hinojosa, Alfredo and Zack, Donald J and Green, Jordan J
Journal: Molecular Therapy (2017)

References


View all 20 references: Citation Explorer
Lectin-histochemical and -cytochemical study of periodic acid Schiff-positive lysosome granules as a histological feature of the female mouse kidney
Authors: Yabuki A, Suzuki S, Matsumoto M, Nishinakagawa H.
Journal: Histol Histopathol (2002): 1017
Alz-50/Gallyas-positive lysosome-like intraneuronal granules in Alzheimer's disease and control brains
Authors: Ikeda K, Akiyama H, Arai T, Kondo H, Haga C, Iritani S, Tsuchiya K.
Journal: Neurosci Lett (1998): 113
The effect of chemical agents on lysosome fusion with phagosomes and on the F-actin content in murine peritoneal macrophages
Authors: Mozhenok TP, Rpozanov Iu M, Solov'eva LV, Braun AD, Bulychev AG.
Journal: Tsitologiia (1992): 84
Autometallography used as a histochemical indicator of lysosome function in cultured cells
Authors: Rungby J, Danscher G, Christensen M, Ellermann-Eriksen S, Mogensen SC.
Journal: Histochemistry (1990): 109
Identification and purification of NK cells with lysosomotropic vital stains: correlation of lysosome content with NK activity
Authors: Shau H, Dawson JR.
Journal: J Immunol (1985): 137
The role of the lysosome in natural killing: inhibition by lysosomotropic vital dyes
Authors: Shau H, Dawson JR.
Journal: Immunology (1984): 745
Alteration in lysosome supravital staining as a marker of hydroxyurea-induced cytotoxicity and its modification by radical scavengers in L5178Y cells in culture
Authors: Grabarczyk M, Przybyszewski WM, Kwiatkowska J, Sitarska E, Malec J.
Journal: Neoplasma (1983): 541
Lysosome changes in exponentially growing, synchronized and differentiating L-cell cultures
Authors: Borisov AB, Bulychev AG, Rumiantsev PP.
Journal: Tsitologiia (1982): 1233
Phorbol myristate acetate stimulates microtubule and 10-nm filament extension and lysosome redistribution in mouse macrophages
Authors: Phaire-Washington L, Silverstein SC, Wang E.
Journal: J Cell Biol (1980): 641
Lysosome stability during lytic infection by simian virus 40
Authors: Einck KH, Norkin LC.
Journal: Intervirology (1979): 47