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Cell Navigator® Lysosome Staining Kit *Deep Red Fluorescence*

Image of Hela cells stained with the A: Cell Navigator® Lysosomal Staining Kit or B: LysoTracker® Red DND99 (from Invitrogen) in a Costar black 96-well plate. The fluorescence signals were compared at 0 and 120 seconds exposure time by using an Olympus fluorescence microscope with Texas red filter set.
Image of Hela cells stained with the A: Cell Navigator® Lysosomal Staining Kit or B: LysoTracker® Red DND99 (from Invitrogen) in a Costar black 96-well plate. The fluorescence signals were compared at 0 and 120 seconds exposure time by using an Olympus fluorescence microscope with Texas red filter set.
 Image of Hela cells stained with LysoBrite™ Deep Red.
Ordering information
Price ()
Catalog Number22659
Unit Size
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Additional ordering information
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)597
Emission (nm)619
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Excitation (nm)
597
Emission (nm)
619
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™ Deep 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

ExcitationTexas Red filter
EmissionTexas Red filter
Recommended plateBlack wall/clear bottom

Components


Component A: LysoBrite™ Deep Red1 vial (50 µL-500X DMSO stock solution)
Component B: Staining Buffer A1 bottle (25 mL)
Component C: Staining Buffer B1 bottle (25 mL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells
  2. Add Lysolite™ Deep Red working solution
  3. Incubate at 37°C for 30 - 60 minutes
  4. Add Staining Buffer B
  5. Incubate at room temperature for 15 - 30 minutes
  6. Analyze the cells under fluorescence microscope at Ex/Em = 590/620 nm (Texas Red filter set)

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

PREPARATION OF WORKING SOLUTION

Add 10 µL of 500X Lysolite™ Deep Red (Component A) into 5 mL of Live Cell Staining Buffer A (Component B) and mix well to make Lysolite™ Deep Red working solution. Protect from light. Note: 10 µL of 500X Lysolite™ Deep 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 1/2 volume (such as 50 µL/well/96-well plate) of Lysolite™ Deep Red working solution.

  3. Incubate the cells in a 37°C, 5% CO2 incubator for 30 to 60 minutes.

  4. Add 50 µL/well (96-well plate) of Staining Buffer B (Component C) into Lysolite™ Deep Red working solution plate.

  5. Incubate at room temperature for 15 - 30 minutes.

  6. Observe the cells using a fluorescence microscope with Texas Red filter set (Ex/Em = 590/620 nm). Note: The LysoBrite™ Deep Red has minimal or no cell toxicity; it can be used for cell tracking. For cell tracking purpose, skip adding Staining Buffer B: Simply replace the dye-working solution with growth medium, and then observe under fluorescence microscope. 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 1000 rpm for 5 minutes to obtain a cell pellet and aspirate the supernatant.

  2. Resuspend the cell pellet gently in pre-warmed growth medium (such as 100 µL/tube), and then add 1/2 volume (50 µL/tube) of Lysolite™ Deep Red working solution.

  3. Incubate the cells in a 37°C, 5% CO2 incubator for 30 to 60 minutes.

  4. Add 50 µL/tube of Staining Buffer B (Component C) into the Lysolite™ Deep Red working solution plate.

  5. Incubate at room temperature for 15 - 30 minutes.

  6. Observe the cells using a fluorescence microscope with Texas red filter set (Ex/Em = 590/620 nm). Note: The LysoBrite™ Deep Red has minimal or no cell toxicity; it can be used for cell tracking. For cell tracking purpose, skip adding Staining Buffer B: Simply replace the dye-working solution with growth medium, and then observe under fluorescence microscope. 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


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spectrum

Spectral properties

Excitation (nm)597
Emission (nm)619

Citations


View all 14 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
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)
Autophagy proteins are not universally required for phagosome maturation
Authors: Cemma, Marija and Grinstein, Sergio and Brumell, John H
Journal: Autophagy (2016): 1440--1446
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
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
Authors: Okoshi, Tadakazu and Yamaguchi, Itaru and Ozawa, Daisaku and Hasegawa, Kazuhiro and Naiki, Hironobu
Journal: PloS one (2015): e0139330

References


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Lectin-histochemical and -cytochemical study of periodic acid Schiff-positive lysosome granules as a histological feature of the female mouse kidney
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Journal: Histol Histopathol (2002): 1017
Alz-50/Gallyas-positive lysosome-like intraneuronal granules in Alzheimer's disease and control brains
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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
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Journal: Tsitologiia (1992): 84
Autometallography used as a histochemical indicator of lysosome function in cultured cells
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Identification and purification of NK cells with lysosomotropic vital stains: correlation of lysosome content with NK activity
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The role of the lysosome in natural killing: inhibition by lysosomotropic vital dyes
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Alteration in lysosome supravital staining as a marker of hydroxyurea-induced cytotoxicity and its modification by radical scavengers in L5178Y cells in culture
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Lysosome changes in exponentially growing, synchronized and differentiating L-cell cultures
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