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LysoBrite™ Red

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. LysoBrite™ Red 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

Assay Protocol with LysoBrite™ Red
  1. Prepare cells.

  2. Add dye working solution.

  3. Incubate at 37 °C for 30 minutes.

  4. Wash the cells.

  5. Analyze under a fluorescence microscope.

Storage and Handling Conditions

The LysoBrite™ Red stock solution provided is 500X in DMSO. It should be stable for at least 6 months if stored at -20°C and protected from light. Avoid freeze/thaw cycles.  

PREPARATION OF WORKING SOLUTION

Prepare LysoBrite™ Red Working Solution
  1. Warm LysoBrite™ Red dye to room temperature.

  2. Prepare dye working solution by diluting 20 µL of 500X LysoBrite™ Red with 10 mL of Hanks and 20 mM HEPES buffer (HBSS) or buffer of your choice.

    Note: 20 µL of LysoBrite™ Red dye is enough for one 96-well plate. Aliquot and store unused LysoBrite™ dye stock solutions at < -15 °C. Protect it from light and avoid repeated freeze-thaw cycles.

    Note: 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. 

SAMPLE EXPERIMENTAL PROTOCOL

This protocol only provides a guideline and should be modified according to your specific needs.

Protocol for Preparing and Staining Adherent Cells
  1. Grow cells 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.

  2. When cells reach the desired confluence, add an equal volume of the dye-working solution (from Preparation of Working Solution Step 2). 

  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. Then fill the cell wells with HBSS or growth medium.

  5. Observe the cells using a fluorescence microscope fitted with the desired 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.

Protocol for Preparing and Staining Suspension Cells
  1. Add an equal volume of the dye-working solution (from Preparation of Working Solution Step 2). 

  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 fitted with the desired 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 treated with BD Cell-Tak® (BD Biosciences) and stained as adherent cells (see Protocol for Preparing and Staining Adherent Cells).

Spectrum

Product family

NameExcitation (nm)Emission (nm)
LysoBrite™ Blue434480
LysoBrite™ Green501510
LysoBrite™ Orange543565
LysoBrite™ Deep Red597619
LysoBrite™ NIR636651

Citations

View all 25 citations: Citation Explorer
Assessing the Effects of Surgical Irrigation Solutions on Human Neutrophil Interactions with Nascent Staphylococcus aureus Biofilms
Authors: Gaur, Gauri and Predtechenskaya, Maria and Voyich, Jovanka M and James, Garth and Stewart, Philip S and Borgogna, Timothy R
Journal: Microorganisms (2024): 1951
In situ reprogramming of tumor-associated macrophages enhances cancer immunotherapy
Authors: Han, Yanjie and Lu, Haifeng and Gu, Zikuan and Guan, Peixin and Liu, Zhen
Journal: Nano Today (2024): 102420
Ferroptosis induces nucleolar stress as revealed by live-cell imaging using thioflavin T
Authors: Hirata, Yoko and Takemori, Hiroshi and Furuta, Kyoji and Kamatari, Yuji O and Sawada, Makoto
Journal: Current Research in Pharmacology and Drug Discovery (2024): 100196
Enhanced Tumor Targeting and Antitumor Activity of Methylated $\beta$-Cyclodextrin-Threaded Polyrotaxanes by Conjugating Cyclic RGD Peptides
Authors: Zhang, Shunyao and Tamura, Atsushi and Yui, Nobuhiko
Journal: Biomolecules (2024): 223
Structural features localizing the ferroptosis inhibitor GIF-2197-r to lysosomes
Authors: Hirata, Yoko and Hashimoto, Tomohiro and Ando, Kaori and Kamatari, Yuji O and Takemori, Hiroshi and Furuta, Kyoji
Journal: RSC Advances (2023): 32276--32281

References

View all 26 references: Citation Explorer
Requirements, features, and performance of high content screening platforms
Authors: Gough AH, Johnston PA.
Journal: Methods Mol Biol (2007): 41
A pharmaceutical company user's perspective on the potential of high content screening in drug discovery
Authors: Hoffman AF, Garippa RJ.
Journal: Methods Mol Biol (2007): 19
Optimizing the integration of immunoreagents and fluorescent probes for multiplexed high content screening assays
Authors: Giuliano KA., undefined
Journal: Methods Mol Biol (2007): 189
Past, present, and future of high content screening and the field of cellomics
Authors: Taylor DL., undefined
Journal: Methods Mol Biol (2007): 3
G protein-coupled receptor internalization assays in the high-content screening format
Authors: Haasen D, Schnapp A, Valler MJ, Heilker R.
Journal: Methods Enzymol (2006): 121
Page updated on November 3, 2024

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Catalog Number22645
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Physical properties

Molecular weight

698.94

Solvent

DMSO

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

Storage

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

Platform

Flow cytometer

Excitation532, 561 nm laser
Emission585, 40 nm filter

Fluorescence microscope

ExcitationTRITC filter set
EmissionTRITC filter set
Recommended plateBlack wall, clear bottom
Images of HeLa cells stained with A: LysoBrite &trade; Red, B: LysoTracker&reg; Red DND-99 (from Invitrogen) in a Costar black wall/clear bottom 96-well plate. Samples were continuously illuminated for 120 seconds, and the signals were compared before and after the exposure by using an Olympus fluorescence microscope.
Images of HeLa cells stained with A: LysoBrite &trade; Red, B: LysoTracker&reg; Red DND-99 (from Invitrogen) in a Costar black wall/clear bottom 96-well plate. Samples were continuously illuminated for 120 seconds, and the signals were compared before and after the exposure by using an Olympus fluorescence microscope.
Images of HeLa cells stained with A: LysoBrite &trade; Red, B: LysoTracker&reg; Red DND-99 (from Invitrogen) in a Costar black wall/clear bottom 96-well plate. Samples were continuously illuminated for 120 seconds, and the signals were compared before and after the exposure by using an Olympus fluorescence microscope.
Lysosome localization and motility is altered for starvation-induced Hela cells. A: Healthy untreated&nbsp; Hela cells. Lysosomes (Red) were dispersed widely throughout the cytosol in cells.&nbsp; 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.
Image of Hela cells stained with LysoBrite™ Red in a Costar black wall-clear bottom 96-well plate.