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

  2. Dilute 20 µL of 500X LysoBrite™ Deep Red with 10 mL of Hanks and 20 mM HEPES buffer (HBSS) or buffer of your choice.

    Note: 20 µL of LysoBrite™ Deep 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™ Red576596
LysoBrite™ NIR636651

Citations

View all 4 citations: Citation Explorer
Through-bond energy transfer-based ratiometric fluorescent probe for the imaging of HOCl in living cells
Authors: Shen, Shi-Li and Ning, Jun-Ya and Zhang, Xiao-Fan and Miao, Jun-Ying and Zhao, Bao-Xiang
Journal: Sensors and Actuators B: Chemical (2017): 907--913
Autophagy proteins are not universally required for phagosome maturation
Authors: Cemma, Marija and Grinstein, Sergio and Brumell, John H
Journal: Autophagy (2016): 1440--1446
Differential detection of tumor cells using a combination of cell rolling, multivalent binding, and multiple antibodies
Authors: Myung, Ja Hye and Gajjar, Khyati A and Chen, Jihua and Molokie, Robert E and Hong, Seungpyo
Journal: Analytical chemistry (2014): 6088--6094
Versatile fabrication of nanoscale sol--gel bioactive glass particles for efficient bone tissue regeneration
Authors: Lei, Bo and Chen, Xiaofeng and Han, Xue and Zhou, Jiaan
Journal: Journal of Materials Chemistry (2012): 16906--16913

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 October 8, 2024

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Catalog Number22646
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Physical properties

Molecular weight

746.99

Solvent

DMSO

Spectral properties

Excitation (nm)

597

Emission (nm)

619

Storage, safety and handling

Certificate of OriginDownload PDF
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

Excitation561 nm laser
Emission630, 30 nm filter

Fluorescence microscope

ExcitationCy3, TRITC filter set
EmissionCy3, TRITC filter set
Recommended plateBlack wall, clear bottom
Image of Hela cells stained with the A: LysoBrite&trade; Deep Redor B: LysoTracker&reg; Red DND-99 (from Invitrogen) in a Costar black 96-well plate. The TRTIC signals were compared at 0 and 120 seconds exposure time by using an Olympus fluorescence microscope.
Image of Hela cells stained with the A: LysoBrite&trade; Deep Redor B: LysoTracker&reg; Red DND-99 (from Invitrogen) in a Costar black 96-well plate. The TRTIC signals were compared at 0 and 120 seconds exposure time by using an Olympus fluorescence microscope.
Image of Hela cells stained with the A: LysoBrite&trade; Deep Redor B: LysoTracker&reg; Red DND-99 (from Invitrogen) in a Costar black 96-well plate. The TRTIC signals were compared at 0 and 120 seconds exposure time by using an Olympus fluorescence microscope.
Image of&nbsp;Hela cells stained with LysoBrite&trade; Deep Red.