logo
AAT Bioquest

Cell Navigator® Lysosome Staining Kit *NIR Fluorescence*

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 NIR fluorescence. LysoBrite™ NIR, 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.

Example protocol

AT A GLANCE

Protocol summary

  1. Prepare cells
  2. Add LysoBrite™ NIR working solution
  3. Incubate at 37°C for 30 minutes
  4. Wash the cells
  5. Analyze the cells under fluorescence microscope at Ex/Em = 630/650 nm (Cy5 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™ NIR stock solution (Component A) to 10 mL of Live Cell Staining Buffer (Component B) to make LysoBrite™ NIR working solution. Protect from light. Note: 20 µL of 500X LysoBrite™ NIR (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. When cells reach the desired confluence, add equal volume of LysoBrite™ NIR working solution.

  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, then fill the cell wells with HBSS or growth medium.

  4. Observe the cells using a fluorescence microscope with Cy5 filter set (Ex/Em = 630/650 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™ NIR 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, then fill the cell wells with HBSS or growth medium.

  4. Observe the cells using a fluorescence microscope with Cy5 filter set (Ex/Em = 630/650 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

Citations

View all 15 citations: Citation Explorer
Biodegradable lipophilic polymeric mRNA nanoparticles for ligand-free targeting of splenic dendritic cells for cancer vaccination
Authors: Ben-Akiva, Elana and Karlsson, Johan and Hemmati, Shayan and Yu, Hongzhe and Tzeng, Stephany Y and Pardoll, Drew M and Green, Jordan J
Journal: Proceedings of the National Academy of Sciences (2023): e2301606120
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
Carboxylated branched poly (β-amino ester) nanoparticles enable robust cytosolic protein delivery and CRISPR-Cas9 gene editing
Authors: Rui, Yuan and Wilson, David R and Choi, John and Varanasi, Mahita and S, undefined and ers, Katie and Karlsson, Johan and Lim, Michael and Green, Jordan J
Journal: Science Advances (2019): eaay3255
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)

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
Page updated on October 12, 2024

Ordering information

Price
Unit size
Catalog Number22652
Quantity
Add to cart

Additional ordering information

Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
Technical SupportContact us
Purchase orderSend to sales@aatbio.com
ShippingStandard overnight for United States, inquire for international
Request quotation

Spectral properties

Excitation (nm)

636

Emission (nm)

651

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200

Platform

Fluorescence microscope

Excitation630 nm
Emission650 nm
Recommended plateBlack wall, clear bottom
Instrument specification(s)Cy5 filter set

Components