Lysosensor yellow blue dnd 160

Manufactured by Thermo Fisher Scientific

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LysoSensor Yellow/Blue DND-160 is a fluorescent probe used for staining and imaging acidic organelles, such as lysosomes, in live cells. It exhibits a pH-dependent shift in fluorescence emission, allowing for the visualization and analysis of lysosomal pH.

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LysoSensor (13 citations) LysoSensor DND-160 (8 citations) LysoSensor Yellow/Blue (8 citations) LysoSensor yellow/blue DND (2 citations) Dextran conjugated Lysosensor Yellow/Blue DND-160 (2 citations) PH sensitive-LysoSensor™ Yellow/Blue DND-160 (2 citations) LysoSensor Yellow/Blue DND-160 dye (2 citations)

91 protocols using lysosensor yellow blue dnd 160

Lysosomal pH Measurement using LysoSensor Dye

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To measure the lysosome’s pH, LysoSensor Yellow/Blue DND-160 (Invitrogen) was used as described previously (Ma et al., 2017 (link)). To determine the pH of lysosomes, a pH calibration curve was first generated for HeLa cells. Briefly, cells were trypsinized and incubated with LysoSensor Yellow/Blue DND-160 (final concentration of 2 µM) in phenol red-free DMEM (Gibco) for 3 min at 37°C. Following incubation, cells were washed twice with DPBS and further incubated with a set of isotonic pH calibration buffers (143 mM KCl, 5 mM Glucose, 1 mM MgCl₂, 1 mM CaCl₂, 20 mM MES, 10 µM Nigericin, and 5 µM monensin) with pH ranging from 4 to 6. Each pH calibration buffer was pre-adjusted to its final pH value using 1 N NaOH or 1 N HCl. Cells (10,000/well) were transferred into a black 96-well plate (Thermo Fisher Scientific), and fluorescence readings were recorded simultaneously for two excitation wavelengths (340 and 380 nm) at 37°C. Finally, the ratio of fluorescence intensity of emission at 340–380 nm against respective pH values was plotted to generate the pH calibration curve. Using this curve, the pH value of lysosomes for HeLa cells treated with indicated siRNA treatment was extrapolated.

Rawat S., Chatterjee D., Marwaha R., Charak G., Kumar G., Shaw S., Khatter D., Sharma S., de Heus C., Liv N., Klumperman J., Tuli A, & Sharma M. (2022). RUFY1 binds Arl8b and mediates endosome-to-TGN CI-M6PR retrieval for cargo sorting to lysosomes. The Journal of Cell Biology, 222(1), e202108001.

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Endo-Lysosomal pH Measurement in BMDMs

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For measurement of endo-lysosomal pH in BMDMs using LysoSensor Yellow/Blue DND-160 (Invitrogen), cells were seeded on black 96-well plates at a density of 100,000 cells per well and cultured for 72h in either untreated complete media or media supplemented with 50nM RSV or PTV:PGRN. Prior to labeling cells, pH calibration solutions were made fresh. pH calibration solutions were generated in 20mM MES (4- Morpholineethanesulfonic acid), 110 mM KCl, and 20mM NaCl, supplemented with 30μM nigericin and 15 μM monensin, sterile filtered, and titrated to pH values of 4.0, 4.5, 5, 5.5, and 6. Cells were labeled with 2uM LysoSensor Yellow/Blue DND-160 in isotonic solution (Hanks Buffered Salt Solution (GIBCO, Life Technologies) supplemented with 60mM mannitol). After 20 min, cells were rinsed 2x with isotonic solution and incubated for 10 min with isotonic solution (test wells) or with pH calibration solutions at 37C/5%CO2. The ratio of light excited at 330nm over 380 nm, detected at a 527 nm emission, was measured at 37 °C using a Spectramax M5 plate reader (Molecular Devices), collected every 2min for 3 cycles and averaged. Absolute pH values were then calculated using a standard curve generated from the pH calibrations, plated in adjacent wells and measured simultaneously.

Logan T., Simon M.J., Rana A., Cherf G.M., Srivastava A., Davis S.S., Yoon Low R.L., Chiu C.L., Fang M., Huang F., Bhalla A., Llapashtica C., Prorok R., Pizzo M.E., Calvert M.E., Sun E.W., Hsiao-Nakamoto J., Rajendra Y., Lexa K.W., Srivastava D.B., van Lengerich B., Wang J., Robles-Colmenares Y., Kim D.J., Duque J., Lenser M., Earr T.K., Nguyen H., Chau R., Tsogtbaatar B., Ravi R., Skuja L.L., Solanoy H., Rosen H.J., Boeve B.F., Boxer A.L., Heuer H.W., Dennis M.S., Kariolis M.S., Monroe K.M., Przybyla L., Sanchez P.E., Meisner R., Diaz D., Henne K.R., Watts R.J., Henry A.G., Gunasekaran K., Astarita G., Suh J.H., Lewcock J.W., DeVos S.L, & Di Paolo G. (2021). Rescue of a lysosomal storage disorder caused by Grn loss-of-function with a brain penetrant progranulin biologic. Cell, 184(18), 4651-4668.e25.

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SFTSV Endosomal pH Measurement in Vero Cells

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The endosomal pH in Vero cells incubated with SFTSV during the entry stage was measured according to the protocol as previously described [13 (link), 31 (link)]. Briefly, Vero cells were incubated with SFTSV (MOI = 1) at 37 °C for 15, 30, 45, or 60 min, and then, the cells were incubated with 25 μM LysoSensor yellow/blue DND-160 (Invitrogen) to label the acidic organelles at 37 °C for 5 min. After washes, the LysoSensor signals in cells were immediately measured at the excitation/emission wavelengths of both 390/435 (blue) and 390/525 nm (green) at each time point. The green/blue ratio was calculated as previously described [13 (link), 31 (link)], and the pH values were determined based on the standard curve between the ratio and pH. The standard curve was established according to a previous study [32 (link)]. Briefly, Vero cells were incubated with LysoSensor yellow/blue for 5 min and then treated with MES buffer (115 × 10⁻³ M KCl, 5 × 10⁻³ M NaCl, 25 × 10⁻³ M MES, and 1.2 × 10⁻⁶ M MgSO₄) of different pH (4.0–7.0) for 10 min. The standard curve between the green/blue ratio and pH values was fitted based on a Boltzmann Sigmoid model in GraphPad Prism (version 7, GraphPad Software, San Diego, CA, USA).

Shen S., Zhang Y., Yin Z., Zhu Q., Zhang J., Wang T., Fang Y., Wu X., Bai Y., Dai S., Liu X., Jin J., Tang S., Liu J., Wang M., Guo Y, & Deng F. (2022). Antiviral activity and mechanism of the antifungal drug, anidulafungin, suggesting its potential to promote treatment of viral diseases. BMC Medicine, 20, 359.

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Lysosomal pH Measurement using LysoSensor

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Measurement of lysosomal pH was determined using dextran conjugates LysoSensor Yellow/Blue DND-160 (Invitrogen, L7545) [26]. In brief, cells were trypsinized, harvested (1 × 10⁶ cells/ml), and loaded with 1 mg/ml LysoSensor-dextran for 1 h at 37°C with 5% CO₂. The cells were then washed 3 times and aliquoted into a black 96-well microplate and pH calibration was performed according to the protocol established by Wolfe et al [26]. In brief, cells were treated with 10 mM monensin (Sigma-Aldrich, M5273) and 10 mM nigericin (Sigma-Aldrich, N7143) in 2-(N-morpholino) ethanesulfonic acid (MES) buffer (5 mM NaCl, 115 mM KCl, 1.3 mM MgSO₄, 25 mM MES [Sigma-Aldrich, M3671]), with the pH adjusted to a range from 3.5–7.0. The samples were read in a Wallac Victor 2 fluorimeter (Perkin Elmer, Norwalk, CT, USA) with excitation at 355 nm. The ratio of emission at 440/535 nm was then calculated for each sample. The pH values were determined from the linear standard curve generated via the pH calibration samples.

Hu W., Zhang L., Li M.X., Shen J., Liu X.D., Xiao Z.G., Wu D.L., Ho I.H., Wu J.C., Cheung C.K., Zhang Y.C., Lau A.H., Ashktorab H., Smoot D.T., Fang E.F., Chan M.T., Gin T., Gong W., Wu W.K, & Cho C.H. (2019). Vitamin D3 activates the autolysosomal degradation function against Helicobacter pylori through the PDIA3 receptor in gastric epithelial cells. Autophagy, 15(4), 707-725.

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Imaging Stimulated Parietal Cell Acidification

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Twelve hours after seeding of parietal cells on coverslips, the medium was replaced by fresh medium. In order to stimulate cells to secrete HCl, they were incubated in medium supplemented with histamine (400 μM; Sigma-Aldrich) and 3-isobutyl-1-methylxanthine (IBMX) (30 μM; Sigma). Control cells were held in a resting state by administering cimetidine (100 μM; Sigma-Aldrich). After 30 min of incubation at 37 °C, cells were incubated in medium A without amphotericin B and supplemented with 2 µM LysoSensor™ Yellow/Blue DND-160 (Invitrogen) and 2 µM Cell Tracker Red CMTPX (Invitrogen) at 37 °C for 30 min. Subsequently, cells were washed 3 times, immediately mounted in a small volume of PBS (50% glycerol, v/v) on glass slides at room temperature, and analyzed using a confocal microscopy within 30 min.

Zhang G., Ducatelle R., Mihi B., Smet A., Flahou B, & Haesebrouck F. (2016). Helicobacter suis affects the health and function of porcine gastric parietal cells. Veterinary Research, 47, 101.

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Ratiometric Lysosome pH Measurement

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Endolysosome pH was measured using a ratio-metric lysosome pH indicator dye (LysoSensor Yellow/Blue DND-160, Invitrogen); a dual excitation dye that permits pH measurements in acidic organelles independently of dye concentration. Neurons were loaded with LysoSensor (2 μM) for 5 minutes at 37°C. Light emitted at 520 nm in response to excitation at 340 nm and 380 nm was measured for 20 ms every 5 seconds using a filter-based imaging system (Zeiss and 3i). The ratios of light excited at 340/380 nm and emitted at 520 nm were converted to pH using a calibration curve established using 10 μM of the H⁺/Na⁺ ionophore monensin, and 20 μM of the H⁺/K⁺ ionophore nigericin dissolved in 20 mM 2-(N-morpholino) ethane sulfonic acid (MES), 110 mM KCl, and 20 mM NaCl adjusted to pH 3.0 to 7.0 with HCl/NaOH.

Hui L., Soliman M.L., Geiger N.H., Miller N.M., Afghah Z., Lakpa K.L., Chen X, & Geiger J.D. (2019). Acidifying endolysosomes prevented LDL-induced amyloidogenesis. Journal of Alzheimer's disease : JAD, 67(1), 393-410.

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Lysosomal pH Measurement Using Ratiometric Dye

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Lysosomal pH was determined using a ratiometric lysosomal pH indicator dye (Lysosensor Yellow/Blue DND-160, Invitrogen) as described [59 (link), 60 (link)].

Frost L.S., Lopes V.S., Bragin A., Reyes-Reveles J., Brancato J., Cohen A., Mitchell C.H., Williams D.S, & Boesze-Battaglia K. (2014). The Contribution of Melanoregulin to Microtubule-Associated Protein 1 Light Chain 3 (LC3) Associated Phagocytosis in Retinal Pigment Epithelium. Molecular neurobiology, 52(3), 1135-1151.

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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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Lysosomal pH Measurement using Lysosensor

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To measure the lysosome’s pH, Lysosensor Yellow/Blue DND-160 was used as described previously^{58 (link)}. Briefly, cells were trypsinized and incubated with 2 µM Lysosensor Yellow/Blue DND-160 (Invitrogen) for 3 min at 37 °C in phenol red-free complete DMEM media. Cells were rinsed twice with 1X PBS to remove excess dye and incubated for 10 min in isotonic pH calibration buffers (143 mM KCl, 5 mM Glucose, 1 mM MgCl₂, 1 mM CaCl₂, 20 mM MES, 10 µM Nigericin, and 5 µM Monensin) ranging from 4 to 6. Next, ~10,000 cells/well were distributed into a black 96-well plate (Thermo Scientific), and fluorescence reading was recorded at 37 °C using a 96-well plate multi-mode fluorescence reader (Tecan Infinite M-PLEX). Samples were excited at 340 and 380 nm wavelengths to detect emitted light at 440 and 540 nm, respectively. The pH calibration curve was generated by plotting the fluorescence intensity ratio of 340 to 380 nm against the respective pH value of buffers.

Kumar G., Chawla P., Dhiman N., Chadha S., Sharma S., Sethi K., Sharma M, & Tuli A. (2022). RUFY3 links Arl8b and JIP4-Dynein complex to regulate lysosome size and positioning. Nature Communications, 13, 1540.

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Lysosomal pH Measurement in HeLa Cells

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Briefly, HeLa cells were cultured in a black-walled, clear-bottomed 96-well plate (PerkinElmer, Inc. USA). After treatment of vehicle, RAP, CQ or 10 μM DAC and DAS for 24 h, the cells were washed and incubated with 5 μM of LysoSensor Yellow/Blue DND-160 (Invitrogen, USA) in complete DMEM without serum for 5 min at 37°C. Light emitted at 535 nm in response to excitation at 340 and 380 nm were determined and the ratio of them was calculated. The ratio was compared to the value of Control and CQ (a classic lysosomal pH neutralizer) treatment.

Wu M.Y., Wang S.F., Cai C.Z., Tan J.Q., Li M., Lu J.J., Chen X.P., Wang Y.T., Zheng W, & Lu J.H. (2017). Natural autophagy blockers, dauricine (DAC) and daurisoline (DAS), sensitize cancer cells to camptothecin-induced toxicity. Oncotarget, 8(44), 77673-77684.

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