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LysoBrite™ Red

Images of HeLa cells stained with A: LysoBrite ™ Red, B: LysoTracker® 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 ™ Red, B: LysoTracker® 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 ™ Red, B: LysoTracker® 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  Hela cells. Lysosomes (Red) were dispersed widely throughout the cytosol in cells.  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.
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Physical properties
Molecular weight698.94
SolventDMSO
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
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200

OverviewpdfSDSpdfProtocol


See also: Lysosomes
Molecular weight
698.94
Excitation (nm)
576
Emission (nm)
596
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.

Platform


Flow cytometer

Excitation532/561 nm laser
Emission585/40 nm filter

Fluorescence microscope

ExcitationTRITC filter set
EmissionTRITC filter set
Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Excitation540
Emission590
Cutoff570
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode

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).

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of LysoBrite™ 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 mM143.074 µL715.369 µL1.431 mL7.154 mL14.307 mL
5 mM28.615 µL143.074 µL286.148 µL1.431 mL2.861 mL
10 mM14.307 µL71.537 µL143.074 µL715.369 µL1.431 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)576
Emission (nm)596

Product Family


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

Images


Citations


View all 22 citations: Citation Explorer
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
Mitochondrial-Targeted Antioxidant MitoQ-Mediated Autophagy: A Novel Strategy for Precise Radiation Protection
Authors: Bao, Xingting and Liu, Xiongxiong and Wu, Qingfeng and Ye, Fei and Shi, Zheng and Xu, Dan and Zhang, Jinhua and Dou, Zhihui and Huang, Guomin and Zhang, Hong and others,
Journal: Antioxidants (2023): 453
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
Haloperidol Prevents Oxytosis/Ferroptosis by Targeting Lysosomal Ferrous Ions in a Manner Independent of Dopamine D2 and Sigma-1 Receptors
Authors: Hirata, Yoko and Oka, Kohei and Yamamoto, Shotaro and Watanabe, Hiroki and Oh-Hashi, Kentaro and Hirayama, Tasuku and Nagasawa, Hideko and Takemori, Hiroshi and Furuta, Kyoji
Journal: ACS Chemical Neuroscience (2022): 2719--2727
Videos of Sipuleucel-T Programmed T Cells Lysing Cells That Express Prostate Cancer Target Antigens
Authors: Kibel, Adam S and Inman, Brant A and Pachynski, Russell K and Vu, Tuyen and Sheikh, Nadeem A and Petrylak, Daniel P
Journal: JNCI: Journal of the National Cancer Institute (2021)
Variable-order fractional master equation and clustering of particles: non-uniform lysosome distribution
Authors: Fedotov, Sergei and Han, Daniel and Zubarev, Andrey Yu and Johnston, Mark and Allan, Victoria J
Journal: arXiv preprint arXiv:2101.02698 (2021)
Delayed Neutrophil Recruitment Allows Nascent Staphylococcus aureus Biofilm Formation and Immune Evasion
Authors: Pettygrove, Brian A and Kratofil, Rachel M and Alhede, Maria and Jensen, Peter {\O} and Newton, Michelle and Qvortrup, Klaus and Pallister, Kyler B and Bjarnsholt, Thomas and Kubes, Paul and Voyich, Jovanka M and others,
Journal: Biomaterials (2021): 120775
Tubeimoside I-induced lung cancer cell death and the underlying crosstalk between lysosomes and mitochondria
Authors: Wang, Kun and Zhan, Yujuan and Chen, Bonan and Lu, Yuhua and Yin, Ting and Zhou, Shikun and Zhang, Weibin and Liu, Xiaodong and Du, Biaoyan and Wei, Xianli and others,
Journal: Cell death \& disease (2020): 1--16
Deciphering anomalous heterogeneous intracellular transport with neural networks
Authors: Han, Daniel and Korabel, Nickolay and Chen, Runze and Johnston, Mark and Gavrilova, Anna and Allan, Victoria J and Fedotov, Sergei and Waigh, Thomas A
Journal: Elife (2020)
17-DMAG disrupted the autophagy flux leading to the apoptosis of acute lymphoblastic leukemia cells by inducing heat shock cognate protein 70
Authors: Xu, Gang and Ma, Xiujuan and Chen, Fang and Wu, Di and Miao, Jianing and Fan, Yang
Journal: Life Sciences (2020): 117532

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