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Cell Navigator® Lysosome Staining Kit *Red Fluorescence*

Images of HeLa cells stained with A: Cell Navigator® Lysosome Staining Kit (Cat# 22658), B: LysoTracker® Red DND-99 (from Invitrogen) in a Costar black wall/clear bottom 96-well plate. The signals were compared at 0 and 120 seconds exposure time by using an Olympus fluorescence microscope.
Images of HeLa cells stained with A: Cell Navigator® Lysosome Staining Kit (Cat# 22658), B: LysoTracker® Red DND-99 (from Invitrogen) in a Costar black wall/clear bottom 96-well plate. The signals were compared at 0 and 120 seconds exposure time by using an Olympus fluorescence microscope.
Image of Hela cells stained with Cell Navigator® Lysosome Staining Kit in a Costar black wall-clear bottom 96-well plate.
Live PDAC microtumours feed on dead-cell debris. (a,b) Time-lapse images of live and dead cell assays. A live PCI-55 microtumour showed that an early apoptotic cell underwent late apoptosis on the surface of microtumours, and was then taken into their bodies (a). PCI-55 microtumours that took up Annexin V<sup>+</sup>/EthD-1<sup>+</sup> cells massively accumulated Annexin V on their surfaces, whereas EthD-1 was dispersed throughout their bodies (b). (c–i) Feeding dead-cell debris to anchorage-dependent PCI-55 microtumours. To maintain PCI-55 microtumours anchored to the micro/nanoplate, we added their UV-induced dead-cell debris. Schematic of the protocol (c). Time-lapse images (d). Time after adding dead-cell debris shown as hh:mm. Grown microtumours devoured dead-cell debris aggressively (e). Tumour sizes in PCI-55 microtumours at 48 h after dead-cell feeding (f–h), CFSE-labelled PCI-55 microtumours were fed UV-induced dead PCI-55 cells into which Edu (red fluorescence) had been incorporated. 3D images of CFSE-labelled PCI-55 microtumours with added Edu<sup>+</sup> dead-cell debris (g). Confocal images of CFSE-labelled PCI-55 microtumours with added Edu<sup>+</sup> dead-cell debris. Cross sections of CFSE-labelled PCI-55 microtumours taking up debris-derived Edu (h). Fluorescence for indicated markers of PCI-55 microtumours taking up debris-derived Edu (i). Zoomed-in image shows that Edu<sup>+</sup> dead-cell debris was endocytosed from the surface of the PDAC microtumour and then incorporated into lysosomes in the inner cells forming the microtumours. Source: <strong>Visualising the dynamics of live pancreatic microtumours self-organised through cell-in-cell invasion</strong> by Miyatake et al., <em>Scientific Reports</em>, Sept. 2018.
Cell Navigator Lysosomal Stain
Lysosome localization and motility is altered for starvation-induced Hela cells. A: Healthy untreated  Hela cells. Lysosomes (Red) were dispersed widely throughout the cytosol in cells.  B: Starved Hela Cells. The cells were starved for 24 hours (no serum), and lysosomes were aggregated in the perinuclear region. Nuclei were stained with Hoechst 33342.
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Catalog Number22658
Unit Size
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Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Spectral properties
Excitation (nm)576
Emission (nm)596
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Excitation (nm)
576
Emission (nm)
596
Lysosomes are cellular organelles which contain acid hydrolase enzymes to break up waste materials and cellular debris. Lysosomes digest excess or worn-out organelles, food particles, and engulfed viruses or bacteria. The membrane around a lysosome allows the digestive enzymes to work at pH 4.5. The interior of the lysosomes is acidic (pH 4.5-4.8) compared to the slightly alkaline cytosol (pH 7.2). The lysosome maintains this pH differential by pumping protons from the cytosol across the membrane via proton pumps and chloride ion channels. Our Cell Navigator® fluorescence imaging kits are a set of fluorescence imaging tools for labeling sub-cellular organelles such as membranes, lysosomes, mitochondria, 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 orange fluorescence. LysoBrite™ Red, the proprietary lysotropic dye used in the kit, selectively accumulates in lysosomes probably via 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 reduces its staining background and makes it useful for a variety of studies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. It is suitable for proliferating and non-proliferating cells, and can be used for both suspension and adherent cells. LysoBrite™ dyes significantly outperform the equivalent LysoTracker ™dyes (from Invitrogen). LysoBrite™ dyes can stay in live cells for more than a week with very minimal cell toxicity while the LysoTracker dyes can only be used for a few hours. LysoBrite™ dyes can survive a few generations of cell division. In addition, LysoBrite™ dyes are much more photostable than the LysoTracker dyes.

Platform


Fluorescence microscope

ExcitationTRITC filter
EmissionTRITC filter
Recommended plateBlack wall/clear bottom

Components


Component A: LysoBrite™ Red1 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™ Red working solution
  3. Incubate at 37°C for 30 minutes
  4. Wash the cells
  5. Analyze the cells under fluorescence microscope at Ex/Em = 575/600 nm (TRITC 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™ Red (Component A) to 10 mL of Live Cell Staining Buffer (Component B) to make LysoBrite™ Red working solution. Protect from light. Note: 20 µL of 500X LysoBrite™ Red (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 cover-slips inside a petri dish filled with the appropriate culture medium.

  2. When cells reach the desired confluence, add equal volume of LysoBrite™ Red working solution.

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

  4. Wash the cells twice with pre-warmed (37°C) Hanks and 20 mM Hepes buffer (HBSS) or buffer of your choice, fill the cell wells with HBSS or growth medium.

  5. Observe the cells using a fluorescence microscope with TRITC filter set (Ex/Em = 575/600 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. Add equal volume of LysoBrite™ Red working solution into the cells.

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

  3. Wash the cells twice with pre-warmed (37°C) Hanks and 20 mM Hepes buffer (HBSS) or buffer of your choice, fill the cell wells with HBSS or growth medium.

  4. Observe the cells using a fluorescence microscope with TRITC filter set (Ex/Em = 575/600 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 cover-slips that have been treated with BD Cell-Tak® (BD Biosciences) and stained as adherent cells.

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)576
Emission (nm)596

Citations


View all 20 citations: Citation Explorer
A surface charge dependent enhanced Th1 antigen-specific immune response in lymph nodes by transfersome-based nanovaccine-loaded dissolving microneedle-assisted transdermal immunization
Authors: Wu, Xuanjin and Li, Yang and Chen, Xiguang and Zhou, Zhongzheng and Pang, Jianhui and Luo, Xin and Kong, Ming
Journal: Journal of Materials Chemistry B (2019): 4854--4866
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
Visualising the dynamics of live pancreatic microtumours self-organised through cell-in-cell invasion
Authors: Miyatake, Yukiko and Kuribayashi-Shigetomi, Kaori and Ohta, Yusuke and Ikeshita, Shunji and Subagyo, Agus and Sueoka, Kazuhisa and Kakugo, Akira and Amano, Maho and Takahashi, Toshiyuki and Okajima, Takaharu and others, undefined
Journal: Scientific reports (2018): 14054
A multi-responsive biomimetic nano-complex platform for enhanced gene delivery
Authors: Bai, Xiaoyu and Kong, Ming and Wu, Xuanjin and Feng, Chao and Park, Hyunjin and Chen, Xiguang
Journal: Journal of Materials Chemistry B (2018): 5910--5921
Chitosan based nanogels stepwise response to intracellular delivery kinetics for enhanced delivery of doxorubicin
Authors: Zuo, Yajun and Kong, Ming and Mu, Yuzhi and Feng, Chao and Chen, Xiguang
Journal: International journal of biological macromolecules (2017): 157--164
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
Vectorization of ultrasound-responsive nanoparticles in placental mesenchymal stem cells for cancer therapy
Authors: Paris, Juan L and de la Torre, Paz and Cabanas, M Victoria and Manzano, Miguel and Grau, Montserrat and Flores, Ana I and Vallet-Reg{\'\i}, Mar{\'\i}a
Journal: Nanoscale (2017): 5528--5537
siRNA-loaded poly (histidine-arginine) 6-modified chitosan nanoparticle with enhanced cell-penetrating and endosomal escape capacities for suppressing breast tumor metastasis
Authors: Sun, Ping and Huang, Wei and Kang, Lin and Jin, Mingji and Fan, Bo and Jin, Hongyan and Wang, Qi-Ming and Gao, Zhonggao
Journal: International journal of nanomedicine (2017): 3221
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)
Statin decreases Helicobacter pylori burden in macrophages by promoting autophagy
Authors: Liao, Wei-Chih and Huang, Mei-Zi and Wang, Michelle Lily and Lin, Chun-Jung and Lu, Tzu-Li and Lo, Horng-Ren and Pan, Yi-Jiun and Sun, Yu-Chen and Kao, Min-Chuan and Lim, Hui-Jing and others,
Journal: Frontiers in cellular and infection microbiology (2017): 203

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