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LysoBrite™ NIR

Image of HeLa cells stained with LysoBrite™ NIR in a Costar black wall/clear bottom 96-well plate.
Image of HeLa cells stained with LysoBrite™ NIR in a Costar black wall/clear bottom 96-well plate.
Image of HeLa cells stained with LysoBrite™ NIR in a Costar black wall/clear bottom 96-well plate.
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Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
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ShippingStandard overnight for United States, inquire for international
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Physical properties
Molecular weight730.89
SolventDMSO
Spectral properties
Excitation (nm)636
Emission (nm)651
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


See also: Lysosomes
Molecular weight
730.89
Excitation (nm)
636
Emission (nm)
651
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™ NIR 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.

Platform


Flow cytometer

Excitation640 nm laser
Emission660/20 nm filter
Instrument specification(s)APC channel

Fluorescence microscope

ExcitationCy5 filter set
EmissionCy5 filter set
Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Excitation620
Emission660
Cutoff630
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode

Example protocol


AT A GLANCE

Assay Protocol with LysoBrite™ NIR
  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™ NIR 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™ NIR Working Solution
  1. Warm LysoBrite™ NIR dye to room temperature.

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

    Note: 20 µL of LysoBrite™ NIR dye is enough for one 96-well plate. Aliquot and store any unused LysoBrite™ dye stock solution at < -15 °C, protected from light. 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™ NIR 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 mM136.819 µL684.097 µL1.368 mL6.841 mL13.682 mL
5 mM27.364 µL136.819 µL273.639 µL1.368 mL2.736 mL
10 mM13.682 µL68.41 µL136.819 µL684.097 µL1.368 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)636
Emission (nm)651

Product Family


NameExcitation (nm)Emission (nm)
LysoBrite™ Blue434480
LysoBrite™ Green501510
LysoBrite™ Orange543565
LysoBrite™ Red576596
LysoBrite™ Deep Red597619

Images


Citations


View all 10 citations: Citation Explorer
Combining single-cell tracking and omics improves blood stem cell fate regulator identification
Authors: Wehling, Arne and Loeffler, Dirk and Zhang, Yang and Kull, Tobias and Donato, Cinzia and Szczerba, Barbara and Camargo Ortega, Germ{\'a}n and Lee, Minkyoung and Moor, Andreas and G{\"o}ttgens, Bertie and others,
Journal: Blood, The Journal of the American Society of Hematology (2022): 1482--1495
Engineering Cardiac Small Extracellular Vesicle-Derived Vehicles with Thin-Film Hydration for Customized microRNA Loading
Authors: Bheri, Sruti and Kassouf, Brandon P and Park, Hyun-Ji and Hoffman, Jessica R and Davis, Michael E
Journal: Journal of Cardiovascular Development and Disease (2021): 135
Antitumor Activity of $\alpha$-Linolenic Acid-Paclitaxel Conjugate Nanoparticles: In vitro and in vivo
Authors: Xu, Mei-Qi and Hao, Yan-Li and Wang, Jing-Ru and Li, Zhuo-Yue and Li, Hui and Feng, Zhen-Han and Wang, Hui and Wang, Jing-Wen and Zhang, Xuan
Journal: International Journal of Nanomedicine (2021): 7269
siRNA Packaged with Neutral Cytidinyl/Cationic/PEG Lipids for Enhanced Antitumor Efficiency and Safety In Vitro and In Vivo
Authors: Zhou, Xinyang and Pan, Yufei and Li, Zheng and Li, Huantong and Wu, Jing and Ma, Yuan and Guan, Zhu and Yang, Zhenjun
Journal: ACS Applied Bio Materials (2020): 6297--6309
Daurisoline inhibits hepatocellular carcinoma progression by restraining autophagy and promoting cispaltin-induced cell death
Authors: Xue, Legang and Liu, Pei
Journal: Biochemical and Biophysical Research Communications (2020)
Anti-tumour activity of low molecular weight heparin doxorubicin nanoparticles for histone H1 high-expressive prostate cancer PC-3M cells
Authors: Zhang, Shuang and Li, Zhan-Tao and Liu, Man and Wang, Jing-Ru and Xu, Mei-Qi and Li, Zhuo-Yue and Duan, Xiao-Chuan and Hao, Yan-Li and Zheng, Xiu-Chai and Li, Hui and others, undefined
Journal: Journal of Controlled Release (2018)
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
Daniel S Han1, 3, 4, Nickolay Korabel1, Runze Chen2, Mark Johnston3, Viki J. Allan3, 4
Authors: Fedotov, Sergei and Waigh, Thomas A

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.
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