Lysotracker dnd 99

Manufactured by Thermo Fisher Scientific

Sourced in United States

LysoTracker DND-99 is a fluorescent dye used to label and visualize acidic organelles, specifically lysosomes, within live cells. It is a membrane-permeant probe that accumulates in low pH environments.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (5 mentions) Fluorescent microscope, by Leica (3 mentions) Cresyl violet, by Merck Group (3 mentions) Csu 22 spinning disk confocal, by Leica (3 mentions) Mitotracker cmxros, by Thermo Fisher Scientific (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions) Lsm 780, by Zeiss (2 mentions) Dq green bsa, by Thermo Fisher Scientific (2 mentions) Fitc dextran 150 kd, by Merck Group (2 mentions) Zymosan beads 568, by Thermo Fisher Scientific (2 mentions) Fl 1301 2, by Vector Laboratories (2 mentions) Hoechst, by Thermo Fisher Scientific (2 mentions) Ib 80827, by Ibidi (2 mentions) Fitc dextran 4kd, by Merck Group (2 mentions) A1r inverted confocal microscope, by Nikon (2 mentions) Fluoromount mounting medium, by Thermo Fisher Scientific (2 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions) Planapo 60 1.42 oil objective, by Olympus (1 mentions) Alexa 647, by Thermo Fisher Scientific (1 mentions) Fluoview 1000 inverted microscope, by Olympus (1 mentions)

Close spelling variants of this product

Lysotracker Red (LTR DND-99; (3 citations) LysoTracker Red DND-99 probe (5 citations) LysoTracker Red DND-99 (961 citations) LysoTracker DS Red DND-99 (2 citations) LysoTracker Red DND-99 solution (2 citations) LysoTracker Red DND-99 dye (13 citations) Lysotracker DND-99 (L-7528) (2 citations) LysoTracker Red DND-99 (L-7528) (9 citations) LysoTracker (310 citations)

39 protocols using lysotracker dnd 99

Lysosomal Activity Quantification by Flow Cytometry and Microscopy

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Cells were incubated with LysoTracker DND-99 (100 nM; Thermo Fisher, Waltham, Massachusetts) during 1 hr at 37°C. Cells were then fixed with 4% paraformaldehyde at room temperature (RT) for 1 hr. Fluorescence was analyzed using a CytoFLEX Flow Cytometer (Beckman Coulter, Brea, California). More than 10,000 events per sample were recorded. The analysis was performed using the FlowJo software.
LysoTracker staining was also analyzed using a Leica TCS SP5 Confocal System. Briefly, cells were washed twice with PBS after incubation with LysoTracker DND-99 (1 µM), fixed with 4% paraformaldehyde for 1 hr at RT, stained with DAPI (1 µg/mL, Thermo Fisher) during 10 min mounted on a glass slide using Fluoromount mounting medium (Thermo Fisher). Quantification of LysoTracker staining was performed using Icy software.

Giraud-Gatineau A., Coya J.M., Maure A., Biton A., Thomson M., Bernard E.M., Marrec J., Gutierrez M.G., Larrouy-Maumus G., Brosch R., Gicquel B, & Tailleux L. (2020). The antibiotic bedaquiline activates host macrophage innate immune resistance to bacterial infection. eLife, 9, e55692.

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Visualizing IgG1 Trafficking in FcRn-expressing Cells

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HMEC1-hFcRn-EGFP cells were seeded in eight-well NuncTM Lab-Tek^TM chambered Coverglass imaging dishes (ThermoFisher Scientific), and grown to 50–70% confluency. WT and IHH hIgG1 variants were conjugated with Alexa-647 following the manufacturer’s procedure (Life Technologies). Cells were washed three times with HBSS pH 6.0 and Alexa-647 conjugated antibodies, diluted in HBSS pH 6.0 to the final concentration of 400 nM, were added to the cells and incubated for 4 h before pictures were taken. The cells were then washed three times with HBSS pH 7.4, and incubated in HBSS pH 7.4 for 4 h before pictures were taken. Lysotracker DND-99 (Life Technologies) was added to the cells 30 min before pictures were taken. Confocal images were acquired on an Olympus FluoView 1000 inverted microscope equipped with a PlanApo 60/1.42 oil objective (Olympus). Constant temperature was set to 37 °C and CO₂ to 5% by an incubator enclosing the microscope stage. Image acquisition was done by sequential line scanning to eliminate bleed-through. Images were processed and prepared with ImageJ (NIH), Adobe Photoshop and Illustrator (Adobe system Inc). Co-localization was quantified using Imaris spot co-localization software (Bitplane) where the endosome size was set to 1 µm. Data from two independent experiments with 15–20 cells analysed were used for co-localization analysis.

Grevys A., Nilsen J., Sand K.M., Daba M.B., Øynebråten I., Bern M., McAdam M.B., Foss S., Schlothauer T., Michaelsen T.E., Christianson G.J., Roopenian D.C., Blumberg R.S., Sandlie I, & Andersen J.T. (2018). A human endothelial cell-based recycling assay for screening of FcRn targeted molecules. Nature Communications, 9, 621.

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Osteoclastogenesis Regulation by WDFY3

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All cell incubations were performed in culture medium consisting of αMEM with 2mM L-glutamine, 10% heat-inactivated FBS, 100 IU/ml Penicillin and 100 IU/ml Streptomycin (Life Technologies, 10007D). Mouse soluble RANKL (R&D Systems, 462TR) and M-CSF ELISA (R&D Systems, DY416), were used for in vitro experiments. CMG14-12 (CMG) media was generated as described before [22 ]. Anti-Wdfy3 (Abnova, clone 2F12), anti-WDFY3 (Novus, NBP1-03332), anti-SQSTM1 (Progen, GP62-C), LC3 antibody (Novus, NB100-2220), anti-β-actin antibody (Cell Signaling, 4970), 800 or 680 secondary antibodies (Li-Cor) were used for in vitro experiments. Cyto-ID kit (Enzo biochem, ENZ-51031) was used for autophagosome staining. LysoTracker DND-99 was used for lysosome staining (Life Technologies, L-7528).

Wu D.J, & Adamopoulos I.E. (2017). Loss of WDFY3 Ameliorates Severity of Serum Transfer-Induced Arthritis Independently of Autophagy. Cellular immunology, 316, 61-69.

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Nanoparticle Uptake Visualization

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For confocal microscopy studies, RAW 264.7 cells were seeded at 10,000 cells/well in an 8-well culture slide (Lab-Tek) and maintained at 37 °C. After adhering, fluorescein-labeled nanoparticles at a final concentration of 1.5 mg/mL in culture media were added to the wells and allowed to incubate for 4 h or 24 h at 37 °C. Culture media used in nanoparticle experiments contained FBS (as indicated in the cell culture section) unless otherwise noted. After incubation, cells were washed with media to remove unbound nanoparticles and then stained with lysotracker DND-99 (Life Technologies, Grand Island, NY) according to manufacturer instructions, in order to confirm endosomal/lysosomal uptake of the nanoparticles. Following staining, cells were imaged with Olympus FV1000 confocal microscopy (Center Valley, PA). Semi-quantitative fluorescence measurements were taken using ImageJ to determine total fluorescent area and intensity of the green channel normalized to the red channel within each field of view (n=3). Results of the PEGylated and non-PEGylated hollow and solid nanoparticles were then compared to determine relative nanoparticle uptake rates.

McMasters J., Poh S., Lin J.B, & Panitch A. (2017). Delivery of Anti-inflammatory Peptides from Hollow PEGylated Poly(NIPAM) Nanoparticles Reduces Inflammation in an Ex Vivo Osteoarthritis Model. Journal of controlled release : official journal of the Controlled Release Society, 258, 161-170.

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Confocal Microscopy of Nanoparticle Uptake in RAW 264.7 Cells

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For confocal microscopy studies, RAW 264.7 cells were seeded in 8 wells culture plates and maintained at 37 °C. Samples were performed in triplicate. RAW 264.7 cells incubated with fluorescein labeled nanoparticles were incubated for 4 h or 24 h at 37 °C in culture medium. Culture media used in nanoparticle experiments contained FBS (as indicated in the cell culture section) unless otherwise noted. After washing with medium to remove any unbound nanoparticles, the cells were imaged using confocal microscopy (Olympus BH-2). Cell samples were subjected to the trypan blue dye exclusion method to check cell viability. Lysotracker DND-99 (Life Technologies, Grand Island, NY, USA) staining was performed according to the manufacturer’s protocol to confirm that nanoparticles were taken up into endosomal/lysosomal intracellular compartments. Semi-quantitative fluorescence measurements were used to compare uptake between PEGylated and non-PEGylated nanoparticles. Image analysis by ImageJ determined the number of fluorescent pixels (area) and the integrated fluorescence intensity for the green channel normalized to the red channel within each field of view (n = 3).

Poh S., Lin J.B, & Panitch A. (2015). Release of anti-inflammatory peptides from thermosensitive nanoparticles with degradable cross-links suppresses pro-inflammatory cytokine production. Biomacromolecules, 16(4), 1191-1200.

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Visualizing Lysosomal Dynamics

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The acid vesicles were labeled with LysoTracker DND-99 (Life Technologies, Molecular Probes) using cells cultured overnight on 35 mm glass bottom culture dishes (MatTek Corporation, Ashland, MA). The cells were rinsed in PBS, incubated with 75 nM of LysoTracker DND-99 in DMEM for 30 minutes. The cells were rinsed with PBS and treated or not with 0.5% 1-butanol or 0.5% tert-butanol. The cells were examined with a Leica TCS-SP5 scanning confocal microscope (Leica).

Brito de Souza L., Pinto da Silva L.L., Jamur M.C, & Oliver C. (2014). Phospholipase D Is Involved in the Formation of Golgi Associated Clathrin Coated Vesicles in Human Parotid Duct Cells. PLoS ONE, 9(3), e91868.

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Real-time Imaging of NK Cell Cytotoxicity

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Target cells were labeled with DiD cell labeling solution (Life Technologies) according to manufacturer instructions and cultured on poly-l-lysine (Sigma-Aldrich) coated Delta T dishes (Bioptechs Inc.) for 1 h at 37°C in phenol free RPMI supplemented with 10% FBS. NK92.p46.GFP effector cells were loaded with 1 μM of LysoTracker DND-99 (Life Technologies) for 30 min, washed, and re-suspended in phenol free RPMI medium containing 200 IU/mL IL-2. Target cells were placed on a heated Delta T stage adapter with a heated lid, and time-lapse images (6 frames/min) of random fields containing target cells were acquired. After imaging had commenced, effector cells were introduced at a 2:1 effector:target ratio. Images were collected on a Nikon Eclipse Ti fluorescence microscope equipped with a CSU-X1 spinning disk confocal head (Yokogawa), Nikon 100X oil objective (NA 1.4), and an XR/MEGA-10 intensified CCD camera (Stanford Photonics). The system was controlled with Micro-Manager software (44 (link)). Nikon Perfect Focus was used to correct drift during time-lapse acquisition.

Hadad U., Thauland T.J., Martinez O.M., Butte M.J., Porgador A, & Krams S.M. (2015). NKp46 Clusters at the Immune Synapse and Regulates NK Cell Polarization. Frontiers in Immunology, 6, 495.

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Visualizing smORF Peptide Localization

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S2 cells were cultured in 10% FBS (v/v) Schneider’s media at 25°C to a confluent stage. Transfections and staining protocols were carried out as described in Pueyo et al. (2016b) (link). We used primary rabbit and mouse anti-Flag antibody (1:500; Sigma) and secondary Donkey anti-mouse FITC (1:1000; Jackson ImmunoResearch), anti-mouse Rhodamine (1:400;Jackson ImmunoResearch) and anti-rabbit Cy5 (1:400; Jackson ImmunoResearch) for detection of tagged-smORF peptides. For detection of mitochondria S2 cells were incubated in 500 nM Mitotracker Red CMXRos (Life Technologies) for 45 min and then fixed for immunohistochemistry. For Lysosome colocalization experiments, cells were incubated in Lysotracker-DND99 (1:1000; Molecular Probes) for 15 min, then mounted and observed in vivo. Images of 5–10 cells per transfection culture were captured on the LSM510 Axioskop 2 or on the Leica TCS SP8 confocal microscopes. For colocalization analysis Z-stack images were taken and analysed with the ImageJ plugin “Mander’s Coefficients” which was used to calculate Pearson’s correlation coefficient of tag to tag signal in 2 different channels.

Pueyo J.I., Salazar J., Grincho C., Berni J., Towler B.P, & Newbury S.F. (2023). Purriato is a conserved small open reading frame gene that interacts with the CASA pathway to regulate muscle homeostasis and epithelial tissue growth in Drosophila. Frontiers in Cell and Developmental Biology, 11, 1117454.

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Immunofluorescence Microscopy for Autophagy

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For IF microscopy cells were plated on poly-L-lysine coverslips (1,5-2 x10⁵ cells /slide). When the appropriate treatments were finished, slides were washed once with PBS, fixed with 4% PFA for 20mins (RT) re-washed twice with PBS, refixed and permeabilized with 100% ice cold MetOH for 10mins (RT) and blocked with 2% BSA in 0,1% saponin (BS). Primary antibodies were left for 1hr at RT or O/N at 4°C. Slides were washed several times with BS and the secondary antibodies were incubated for 1hr at RT. Antibodies used were mouse anti-LC3B (1:20, 5F10 nanoTools), rat anti-Lamp-1 (1:400, 1D4B Abcam), rabbit anti-p62 (1:500, MBL), rabbit anti-TFEB (1:100, 954604 R&D Systems) followed by incubation with Alexa fluor® 555 anti-mouse IgG (1:500, Molecular Probes), Alexa fluor® 488 anti-rabbit IgG (1:500, Molecular Probes), Alexa fluor® 633 anti-rat IgG (1:250, Molecular Probes). For visualization of the nuclei DAPI (Sigma-Aldrich) was used. Samples were mounted with Mowiol and visualized using inverted confocal live cell imaging system Leica SP8.
For Mitotracker CMXRos and Lysotracker DND-99 (Molecular Probes) stainings, dyes were added in culture as suggested by the manufacturer, washed, fixed with 4% PFA for 20mins (RT), and mounted for immediate observation under the microscope.

Gkirtzimanaki K., Kabrani E., Nikoleri D., Polyzos A., Blanas A., Sidiropoulos P., Makrigiannakis A., Bertsias G., Boumpas D.T, & Verginis P. (2018). IFNα Impairs Autophagic Degradation of mtDNA Promoting Autoreactivity of SLE Monocytes in a STING-Dependent Fashion. Cell Reports, 25(4), 921-933.e5.

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Comprehensive Antibody Panel for Autophagy

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Anti-S. pneumoniae (SSI), anti-GFP (Cell Signaling), anti-Myc (9B11, Cell Signaling), anti-Flag (Wako), anti-Galectin-3 (SINO BIOLOGICAL), anti-Calcoco2 (Proteintech for WB), anti-NDP52 (Gene Tex (GTX115378) for IF), anti-K63 linked Ub (clone Apu3, EMD Millipore), anti-LC3, p62, RFP, Atg16L1, ubiquitin (MBL), and anti-actin (Santa Cruz Biotechnology, Inc.) were used as primary antibodies. An HRP-conjugated goat anti-rabbit or anti-mouse antibodies (Jackson Laboratories) were used as secondary antibodies for immunoblotting. FITC- or TRITC-conjugated goat anti-rabbit or anti-mouse IgG antibodies (Sigma-Aldrich) were used as secondary antibodies for immunostaining. DAPI (4′,6-diamidino- 2-phenylindole, Sigma-Aldrich) was used for DNA staining. LysoTracker DND-99 was purchased from molecular probes. 10 µM rapamycin and 40 µM chloroquine (Selleck chemical), and 30 µM PYR-41 (UBPBio), and 10 mM 3-methyladenine (3-MA, Wako) were used as autophagy inducer or inhibitor. 300 µM Apocynin (Selleck), 10 mM GSH (Cayman Chemical), 2.5 mM NAC (Sigma-Aldrich) were used as antioxidative reagents. All other reagents were purchased from Sigma-Aldrich. All antibodies were used at 1:100 for immunofluorescence staining and 1:1000 for western blotting.

Ogawa M., Takada N., Shizukuishi S., Tomokiyo M., Chang B., Yoshida M., Kakuta S., Tanida I., Ryo A., Guan J.L., Takeyama H, & Ohnishi M. (2020). Streptococcus pneumoniae triggers hierarchical autophagy through reprogramming of LAPosome-like vesicles via NDP52-delocalization. Communications Biology, 3, 25.

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