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

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.

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 21 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
Insight into Cellular Uptake and Transcytosis of Peptide Nanoparticles in Spodoptera frugiperda Cells and Isolated Midgut
Authors: McGraw, Erin and Roberts, Jonathan D and Kunte, Nitish and Westerfield, Matthew and Streety, Xavier and Held, David and Avila, L Adriana
Journal: ACS omega (2022): 10933--10943
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

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 11, 2024

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Catalog Number22651
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Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

Platform

Fluorescence microscope

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

Components