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Cell Navigator® Lysosome Staining Kit *Blue Fluorescence*

Image of HeLa cells stained with Cell Navigator® Lysosomal Staining Kit in a Costar black wall/clear bottom 96-well plate.
Image of HeLa cells stained with Cell Navigator® Lysosomal Staining Kit in a Costar black wall/clear bottom 96-well plate.
β2-m amyloid fibrils are internalized and sorted to lysosomes. HIG-82 cells incubated with Ham’s F12 medium containing vehicle buffer, 10 μg/ml β2-m monomer, or 10 μg/ml β2-m fibrils for 12 hrs were stained for lysosomes (red), β2-m (green), and nuclei (blue), and observed with the confocal laser microscope as described in Materials and Methods. When the cells were incubated with fibrils (right column), green fluorescence indicating β2-m fibrils were observed inside the cells in a granular pattern, as well as on the surface of the cells. Importantly, some green-colored granules containing β2-m fibrils were merged with red-colored lysosomes. The scale bars are 10 μm long. *HIG-82 cells cultured on a glass bottom culture dish (P35G-0-14-C, MatTek), were incubated with Ham’s F12 medium containing vehicle buffer, 10 μg/ml β2-m monomer, or 10 μg/ml β2-m fibrils for 12 hrs, washed twice with culture medium, stained with lysotracker (Cell Navigator Lysosome Staining Kit, AAT Bioquest, Inc., Sunnyvale, CA, USA) according to the manufacturer’s instructions, washed twice with culture medium, and fixed with 4% paraformaldehyde in PBS for 30 min at 37°C in the dark. Source: Graph from <strong>Endocytosed β2-Microglobulin Amyloid Fibrils Induce Necrosis and Apoptosis of Rabbit Synovial Fibroblasts by Disrupting Endosomal/Lysosomal Membranes: A Novel Mechanism on the Cytotoxicity of Amyloid Fibrils</strong> by Tadakazu Okoshi, et al., <em>PLoS ONE</em>, Sep.  2015. 
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Catalog Number22655
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Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
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H-phraseH303, H313, H333
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Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
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 blue 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.

Platform


Fluorescence microscope

Excitation360 nm
Emission445 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)DAPI filter set

Components


Component A: LysoBrite™ Blue1 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 dye working solution
  3. Incubate at 37°C for 30 minutes to 2 hours
  4. Analyze under fluorescence microscope at Ex/Em = 360/445 nm (DAPI filter set)

Important notes
Warm LysoBrite™ Blue (Component A) to room temperature.

PREPARATION OF WORKING SOLUTION

Dilute 20 µL of LysoBrite™ Blue (Component A) into 10 mL of Live Cell Staining Buffer (Component B). Protect from light. Note: 20 µL of LysoBrite™ Blue (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

  1. Prepare samples and cells.

    For adherent cells: Grow cells either in a black wall/clear bottom 96-well 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 the dye-working solution.

    For suspension cells: Centrifuge the cells at 1,000 rpm for 5 minutes to obtain a cell pellet and aspirate the supernatant. Resuspend the cell pellet gently in pre-warmed growth medium, and then add equal volume of the dye-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 fitted with a DAPI filter set. 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. Note: 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 15 citations: Citation Explorer
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
New compound ChlA-F induces autophagy-dependent anti-cancer effect via upregulating Sestrin-2 in human bladder cancer
Authors: Hua, Xiaohui and Xu, Jiheng and Deng, Xu and Xu, Jiawei and Li, Jingxia and Zhu, David Q and Zhu, Junlan and Jin, Honglei and Tian, Zhongxian and Huang, Haishan and others,
Journal: Cancer letters (2018): 38--51
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)
Size-dependent mechanism of intracellular localization and cytotoxicity of mono-disperse spherical mesoporous nano-and micron-bioactive glass particles
Authors: Li, Yuli and Hu, Qing and Miao, Guohou and Zhang, Qing and Yuan, Bo and Zhu, Ye and Fu, Xiaoling and Chen, Xiaofeng and Mao, Chuanbin
Journal: Journal of biomedical nanotechnology (2016): 863
Rhodamine bound maghemite as a long-term dual imaging nanoprobe of adipose tissue-derived mesenchymal stromal cells
Authors: Cmiel, Vratislav and Skopalik, Josef and Polakova, Katerina and Solar, Jan and Havrdova, Marketa and Milde, David and Justan, Ivan and Magro, {cmiel2016rhodamine, title={Rhodamine bound maghemite as a long-term dual imaging nanoprobe of adipose tissue-derived mesenchymal stromal cells
Journal: European Biophysics Journal (2016): 1--12
Decidua-derived mesenchymal stem cells as carriers of mesoporous silica nanoparticles. In vitro and in vivo evaluation on mammary tumors
Authors: Paris, Juan L and de la Torre, Paz and Manzano, Miguel and Cabanas, M Victoria and Flores, Ana I and Vallet-Regí, María
Journal: Acta biomaterialia (2016): 275--282
Fluorescence imaging of siRNA delivery by peptide nucleic acid-based probe
Authors: Sato, Takaya and Sato, Yusuke and Iwai, Kenta and Kuge, Shusuke and Teramae, Norio and Nishizawa, Seiichi
Journal: Analytical Sciences (2015): 315--320
The consideration of indolicidin modification to balance its hemocompatibility and delivery efficiency
Authors: Tsai, Ching-Wei and Hu, Wei-Wen and Liu, Chih-I and Ruaan, Ruoh-Chyu and Tsai, Bing-Chang and Jin, Shiow-Lian Catherine and Chang, Yung and Chen, Wen-Yih
Journal: International journal of pharmaceutics (2015): 498--505

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