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LysoBrite™ Deep Red

Image of Hela cells stained with the A: LysoBrite™ Deep Redor B: LysoTracker® 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™ Deep Redor B: LysoTracker® 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 LysoBrite™ Deep Red.
Ordering information
Price ()
Catalog Number22646
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
Physical properties
Molecular weight746.99
SolventDMSO
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
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Molecular weight
746.99
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. 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.

Platform


Fluorescence microscope

ExcitationCy3/Texas Red filter set
EmissionCy3/Texas Red filter set
Recommended plateBlack wall/clear bottom

Example protocol


AT A GLANCE

Protocol summary
  1. Prepare cells
  2. Add LysoBrite™ Deep Red working solution
  3. Incubate at 37 °C for 30 minutes
  4. Wash cells
  5. Analyse using fluorescence microscope with Cy3/Texas Red filter set 

Important
Thaw LysoBrite™ stock solution to room temperature. The LysoBrite™ stock solutions provided are 500X in DMSO. They should be stable for at least 6 months if store at - 20 °C. Protect the fluorescent conjugates from light, and avoid freeze/thaw cycles.

PREPARATION OF WORKING SOLUTION

LysoBrite™ Deep Red working solution
Prepare dye working solution by diluting 20 µL of LysoBrite™ Deep Red (500X) to 10 mL of Hanks and 20 mm Hepes buffer (HHBS) or buffer of your choice.
Note     20 µL of LysoBrite™ 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][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.

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 the LysoBrite™ Deep Red 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 (HHBS) or buffer of your choice, fill the cell wells with HHBS or growth medium.
  4. Observe the cells using a fluorescence microscope with Cy3/Texas Red 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. 

For suspension cells
  1. Add equal volume of LysoBrite™ Deep Red working solution into the cells. Incubate the cells in a 37 °C, 5% CO2 incubator for 30 minutes.
  2. Wash the cells twice with pre-warmed (37 °C) Hanks and 20 mM Hepes buffer (HHBS) or buffer of your choice, fill the cell wells with HHBS or growth medium.
  3. Observe the cells using a fluorescence microscope with Cy3/Texas Red 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 cover-slips that have been treated with BD Cell-Tak® (BD Biosciences) and stained as adherent cells. 

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of LysoBrite™ Deep Red to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM133.871 µL669.353 µL1.339 mL6.694 mL13.387 mL
5 mM26.774 µL133.871 µL267.741 µL1.339 mL2.677 mL
10 mM13.387 µL66.935 µL133.871 µL669.353 µL1.339 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)597
Emission (nm)619

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
High-content fluorescence-based screening for epigenetic modulators
Authors: Martinez ED, Dull AB, Beutler JA, Hager GL.
Journal: Methods Enzymol (2006): 21
Application of laser-scanning fluorescence microplate cytometry in high content screening
Authors: Bowen WP, Wylie PG.
Journal: Assay Drug Dev Technol (2006): 209
High-content screening of known G protein-coupled receptors by arrestin translocation
Authors: Hudson CC, Oakley RH, Sjaastad MD, Loomis CR.
Journal: Methods Enzymol (2006): 63
Evaluation of a high-content screening fluorescence-based assay analyzing the pharmacological modulation of lipid homeostasis in human macrophages
Authors: Werner T, Liebisch G, Gr and l M, Schmitz G.
Journal: Cytometry A (2006): 200
Automated high content screening for phosphoinositide 3 kinase inhibition using an AKT 1 redistribution assay
Authors: Wolff M, Haasen D, Merk S, Kroner M, Maier U, Bordel S, Wiedenmann J, Nienhaus GU, Valler M, Heilker R.
Journal: Comb Chem High Throughput Screen (2006): 339
High concordance of drug-induced human hepatotoxicity with in vitro cytotoxicity measured in a novel cell-based model using high content screening
Authors: O'Brien P J, Irwin W, Diaz D, Howard-Cofield E, Krejsa CM, Slaughter MR, Gao B, Kaludercic N, Angeline A, Bernardi P, Brain P, Hougham C.
Journal: Arch Toxicol (2006): 580