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Lysosensor dnd 189

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LysoSensor DND-189 is a fluorescent dye that selectively accumulates in cellular acidic compartments, such as lysosomes. It is used as a tool for monitoring lysosomal pH and dynamics in living cells.

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Lab products found in correlation

H2dcfda, by Thermo Fisher Scientific (2 mentions) Lysosensor yellow blue dnd 160, by Thermo Fisher Scientific (1 mentions) Myeloperoxidase, by Merck Group (1 mentions) Graphite, by Merck Group (1 mentions) Hydrazine monohydrate, by Merck Group (1 mentions) Chelex 100 resin, by Merck Group (1 mentions) Dq ova, by Thermo Fisher Scientific (1 mentions) C18pmh, by Merck Group (1 mentions) Murine class b cpg odn 1826, by Integrated DNA Technologies (1 mentions) Lysotracker, by Thermo Fisher Scientific (1 mentions) N acetyl l cysteine, by Merck Group (1 mentions) S 2 4 isothiocyanatobenzyl 1 4 7 triazacyclononane 1 4 7 triacetic acid p scn bn nota, by Macrocyclics (1 mentions) Lysosensor green dnd 189, by Thermo Fisher Scientific (1 mentions) Celldiscoverer 7, by Zeiss (1 mentions) Lsm 800, by Zeiss (1 mentions) Annexin 5 apoptosis kit, by BD (1 mentions) Intracellular fixation and permeabilization buffer set kit, by Thermo Fisher Scientific (1 mentions) Mitosox, by Thermo Fisher Scientific (1 mentions) Facscalibur, by BD (1 mentions)

6 protocols using lysosensor dnd 189

1

Lysosensor-based Astrocyte pH Analysis

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For detection of changes in lysosomal pH, astrocytes cultured on poly-L-lysine–coated glass slides were stained with 5 μM Lysosensor DND-189 or Lysosensor Yellow-Blue DND-160 (Invitrogen) dye in growth medium for 30 minutes in a humidified CO2 incubator. The cells were then transferred to Live Cell Imaging Solution and the resulting images were obtained using an LSM780 confocal Live-Cell Imaging System.
Kim H.N., Seo B.R., Kim H, & Koh J.Y. (2020). Cilostazol restores autophagy flux in bafilomycin A1-treated, cultured cortical astrocytes through lysosomal reacidification: roles of PKA, zinc and metallothionein 3. Scientific Reports, 10, 9175.
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2

Lysosomal Staining with LysoSensor DND-189

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LysoSensor™ DND-189 (Invitrogen) has a pKa of 5.2; the compound is fluorescent when located in acidic vesicles, such as lysosomes. Following treatment, growth medium was aspirated, and cells were incubated with 10 μm Hoechst 33342 and labeled with 1 μM LysoSensor™ DND-189 for 20 minutes at 37oC.
Maysinger D., Gran E.R., Bertorelle F., Fakhouri H., Antoine R., Kaul E.S., Samhadaneh D.M, & Stochaj U. (2020). Gold nanoclusters elicit homeostatic perturbations in glioblastoma cells and adaptive changes of lysosomes. Theranostics, 10(4), 1633-1648.
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3

Lysosomal pH Measurement Protocol

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LysoSensor DND-189 (Invitrogen, L7535, USA) was used to measure the lysosomal pH. After treatment with BI6727 for 24 h, NRK49F or mPTC cells were incubated in prewarmed (37 °C) probe-containing medium for 40 min. Then, the probe-containing medium was replaced with loading buffer, and the cells were observed under a fluorescence microscope.
Du Y., Shang Y., Qian Y., Guo Y., Chen S., Lin X., Cao W., Tang X., Zhou A., Huang S., Zhang A., Jia Z, & Zhang Y. (2023). Plk1 promotes renal tubulointerstitial fibrosis by targeting autophagy/lysosome axis. Cell Death & Disease, 14(8), 571.
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4

Multifunctional Nanoparticle Synthesis and Characterization

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All reagents used in this work were analytical or higher grade. Graphite, C18PMH (poly(maleic anhydride-alt-1-octadecene)), hydrazine monohydrate, myeloperoxidase, N-acetyl-l-cysteine, and Chelex 100 resin were purchased from Sigma-Aldrich. MAL-PEG5k-NHS, DSPE-PEG2k-NH2, and Boc-NH-PEG3k-NH2 were purchased from Creative PEGworks (Winston Salem, NC, USA). S-2-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) was purchased from Macrocyclics, Inc. (Dallas, TX, USA). H2DCFDA, Lysotracker, LysoSensor DND 189, and DQ-OVA were purchased from Thermo Fisher Scientific. Antigen peptides, including Adpgk (CSSASMT-NMELM), M27 (LCPGNKYEM), and M30 (CSSVDWEN-VSPELNSTDQ), were synthesized by RS Synthesis (Louisville, KY, USA). Murine class B CpG ODN 1826 was purchased from Integrated DNA Technologies (Coralville, IA, USA). Anti-mouse PD-1 IgG (clone: RMP1–14) was purchased from BioXcell. All other chemicals and reagents were purchased from Thermo Fisher Scientific.
Xu C., Hong H., Lee Y., Park K.S., Sun M., Wang T., Aikins M.E., Xu Y, & Moon J.J. (2020). Efficient Lymph Node-Targeted Delivery of Personalized Cancer Vaccines with Reactive Oxygen Species-Inducing Reduced Graphene Oxide Nanosheets. ACS nano, 14(10), 13268-13278.
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5

Lysosomal Acidification Assay in BMMs

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Lysosomal acidification assay was performed using the IncuCyte pHrodo Red Cell-Labeling kit (Sartorius) or LysoSensor DND-189 (Thermo Fisher). For assay using pHrodo, apoptotic thymocytes were stained with 1 µg ml−1 of pHrodo at 37 °C for 1 h and then with 2.5 µM CellTracker CFSE (Thermo Fisher) at 37 °C for 20 min.
For assays using LysoSensor, 2 h before the assay, BMMs were stained with 1 µM LysoSensor Green DND-189 (Thermo Fisher) following the manufacturer’s protocol. Apoptotic thymocytes were stained with 5 µl ml−1 Vybrant DiD for 45 min at 37 °C in the dark and incubated with BMMs.
Live-cell imaging was performed on a Cell Discoverer 7 or LSM800 microscopes (Zeiss). Peak intensity of pHrodo or LysoSensor was analyzed using Zen v.3.8 (Zeiss) or CellTracker (Warwick University).
Astuti Y., Raymant M., Quaranta V., Clarke K., Abudula M., Smith O., Bellomo G., Chandran-Gorner V., Nourse C., Halloran C., Ghaneh P., Palmer D., Jones R.P., Campbell F., Pollard J.W., Morton J.P., Mielgo A, & Schmid M.C. (2024). Efferocytosis reprograms the tumor microenvironment to promote pancreatic cancer liver metastasis. Nature Cancer, 5(5), 774-790.
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6

Multiparametric Immune Cell Profiling

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Live cells (Annexin V Apoptosis kit, BD PharMingen) were stained with fluorochrome-conjugated antibodies (anti-CD14 clone HCD14, anti-CD4 clone Okt4, anti-CD86 clone IT2.2, anti-HLADR cloneL243, anti - p4EBP1 clone T36/45, anti-pP70S6K clone Cl 215247) for 20 min at 4°C in PBS/5% FBS. Mitochondrial ROS were measured by MitoSOX (Invitrogen) staining (1 mM for 15min at 37°C and 5% CO2). Mitochondrial membrane polarization was assessed by JC-1 (eBiosciences) staining (1 μg/ml for 10min at 37°C and 5% CO2) and lysosomal re-acidification by aNPs was assessed using.Lysosensor DND189 (Thermo Scientific) staining (1μΜ in medium for 20min at 37°C and 5% CO2). Samples were acquired on a FACSCalibur (BD Biosciences) and analyzed using the FlowJo software (Tree Star). For phospho-protein staining, cells were permeabilized and stained using the Intracellular Fixation and Permeabilization Buffer Set kit (eBioscience), according to manufacturer instructions.
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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