Cells were treated as described and labeled with 10 μM Lysosensor Blue DND-167 (Molecular Probes, Eugene, OR) as previously described [10 (link)]. Cells were resuspended in fresh medium prior to examination using an Olympus FV1000 confocal laser scanning microscope system, based on an Olympus IX-81 ZDC microscope, with BP 330-385 nm excitation and BA 420nm emission filters. Images were captured using FV10-ASW 1.7 software and the number of acidic vacuoles in cells quantitated using ImageJ software.
Lysosensor blue dnd 167
Manufactured by Thermo Fisher Scientific
Sourced in United States
LysoSensor Blue DND-167 is a fluorescent probe used for the detection and localization of acidic organelles, such as lysosomes, in living cells. It exhibits a pH-dependent fluorescence, with increased emission in acidic environments. The product is intended for research use only.
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Lab products found in correlation
Lysotracker red dnd 99, by Thermo Fisher Scientific (2 mentions)
Attune nxt, by Thermo Fisher Scientific (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Ix81 zdc microscope, by Olympus (1 mentions)
Fv1000 confocal laser scanning microscope system, by Olympus (1 mentions)
Spe confocal microscope, by Leica (1 mentions)
Fixation permeabilization kit, by BD (1 mentions)
Lamp2, by Abcam (1 mentions)
Prolong gold antifade mountant with dapi nuclear stain, by Thermo Fisher Scientific (1 mentions)
Mouse anti human lamp1, by BD (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Bx61 fluorescent microscope, by Olympus (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Cellrox green, by Thermo Fisher Scientific (1 mentions)
Eclipse ni light microscope, by Nikon (1 mentions)
C11440 digital camera, by Hamamatsu Photonics (1 mentions)
Axiocammr3 camera, by Zeiss (1 mentions)
Rat anti ha, by Merck Group (1 mentions)
Axio imager z2, by Zeiss (1 mentions)
10 protocols using lysosensor blue dnd 167
1
Quantitative Lysosomal Imaging in Cells
2
Quantifying Lysosomal Markers in Cells
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Cells were grown on four-well glass slide (Millipore) in normal growth medium for 24 h. Then the cells were fixed, permeabilized using the fixation/permeabilization kit (BD Biosciences catalogue 554 714) and incubated with primary mouse anti-human LAMP1 (BD Biosciences), LAMP2 (Abcam) or EEA-1 (BD Biosciences) monoclonal antibodies and an Alexa Fluor 488 conjugated goat anti-mouse IgG (H + L) antibody. The slides were mounted using ProLong Gold antifade Mountant with DAPI nuclear stain (ThermoFisher Scientific). The slides were observed on a Leica Sp5 confocal microscope, and ≥10 images each containing ≥3 cells were acquired. Images from three independent experiments were analysed using ImageJ software. The fluorescence intensity per cell was quantified in images of maximum intensity Z-projections. The cellular area, the integrated density and the mean grey values were analysed. Measurements of regions without fluorescence were used for background subtraction. The net average fluorescence intensity per pixel, expressed as corrected total cell fluorescence (CTCF), was calculated for each cell. In addition, the cells were stained with LysoSensor Blue DND-167 1 μM or LysoTracker Red DND-99100 nM (Molecular Probes, ThermoFisher Scientific) for 1 h and observed on a Leica SPE confocal microscope.
3
Lysosomal Visualization in Dictyostelium
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Cells were grown in HL5 medium to a density of 1–3 × 106 cells ml−1. An aliquot containing 106 cells were harvested (1,500 ×g for 2 min) and resuspended in Lo-Flo HL5 containing 500 nM LysoSensor Blue DND-167 (Invitrogen) and incubated at 21°C shaking for 30 min. Cells were washed 2 times in 1 × PBS, resuspended to a density of 1 × 106 cells ml−1 and 10 μl of suspension mounted onto microscope slides. Live cells were viewed through the DAPI filter with an Olympus BX61 fluorescent microscope.
4
Lysosomal Activity Imaging and Analysis
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After the indicated treatments, either LysoTracker Red DND-99 final concentration 25 nM or LysoSensor Blue DND-167 final concentration 10 μM (both Invitrogen) was added and incubated at 37°C for 30 min. For confocal microscopy, the medium was replaced with fresh medium and cells were observed under a Leica TCS SP5 confocal microscope. For flow cytometric analysis, cells were trypsinized and MFI was analyzed with an Attune NxT (Thermo Fisher). A minimum of 10,000 events per sample was acquired.
5
Multimodal Visualization of Cellular Senescence
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At 30 min posttreatment, live dSH cells were stained with 2 μM GlycoGREEN™-βGal (GC611, Goryo Chemical Inc., Hokkaido, Japan), 5 μM 2′,7′-dichlorofluorescein diacetate (DCFDA; 287810, Calbiochem, San Diego, CA, USA), 2 μM LysoSensor™ Blue DND-167 (L7533, Invitrogen, Carlsbad, CA, USA), and 1 μM Hoechst 33342 for examining SA β-gal activity, ROS, active lysosomes, and nucleus, respectively. The stained cells were mounted with ProLong™ Diamond Antifade Mountant (P36965, Invitrogen) and imaged using a confocal laser scanning microscope. Differential interference contrast images were obtained using a confocal laser scanning microscope.
6
Lysosomal Acidification Measurement in Dictyostelium
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Dictyostelium cells were grown in HL5 medium to a density of 1–3 × 106 cells ml−11 × 107 cells were harvested (1,500 ×g for 2 min) and diluted 1:10 in Lo-Flo HL5 either with or without 500 nM LysoSensor™ Blue DND-167 (Invitrogen) and incubated covered in foil at 21°C shaking for 30 min. Cells were washed twice with 1 × PBS and resuspended to a density of 0.5 × 106 cells ml−1 in PBS and the fluorescence of a 2 ml sample measured in a Modulus™ 9200-003 fluorometer (Turner BioSystems, Sunnyvale, CA, United States) using the UV module kit 9200-041 (Ex. 365 nm, Em. 410–460 nm). The background fluorescence of the cells that had not been dyed was subtracted from the fluorescence recorded from the Lysosensor Blue stained cells.
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Dictyostelium cells were grown in HL5 medium to a density of 1–3 × 106 cells ml−11 × 107 cells were harvested (1,500 ×g for 2 min) and diluted 1:10 in Lo-Flo HL5 either with or without 500 nM LysoSensor™ Blue DND-167 (Invitrogen) and incubated covered in foil at 21°C shaking for 30 min. Cells were washed twice with 1 × PBS and resuspended to a density of 0.5 × 106 cells ml−1 in PBS and the fluorescence of a 2 ml sample measured in a Modulus™ 9200-003 fluorometer (Turner BioSystems, Sunnyvale, CA, United States) using the UV module kit 9200-041 (Ex. 365 nm, Em. 410–460 nm). The background fluorescence of the cells that had not been dyed was subtracted from the fluorescence recorded from the Lysosensor Blue stained cells.
7
Fluorescence Microscopy of Malaria Parasites
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The transgenic P. berghei parasite line pv5-tag-GFPPV (27 (link)) was imaged live using an AxioImager Z2 epifluorescence microscope equipped with an AxioCam MR3 camera (Zeiss). For mechanical parasite expansion, 1 to 2 µL of infected blood was incubated under a 22 × 40-mm coverslip for several minutes until erythrocyte lysis became apparent. P. falciparum parasites were imaged on an Eclipse Ni light microscope (Nikon) fitted with a C11440 digital camera (Hamamatsu). Immunofluorescence analysis was performed with P. falciparum pv5-3xHA:loxP parasites that were fixed in 4% formaldehyde using rat anti-HA (1:500; Sigma Aldrich) and rabbit anti-SERA5 (1:500) (55 (link)) primary antibodies in combination with appropriate fluorophore-coupled secondary antibodies (1:1,000; Thermo Fisher Scientific). Staining with Lysosensor blue DND-167 (Thermo Fisher Scientific), BODIPY 581/591 C11 (Image-iT Lipid Peroxidation Kit, Thermo Fisher Scientific), and CellROX Green (Thermo Fisher Scientific) was performed according to the manufacturer’s instructions.
8
Lysosomal pH Regulation Using Bafilomycin A1
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Bafilomycin A1 was obtained from InvivoGen (San Diego, CA, USA). Stock solutions of 100 μM (160 μl DMSO added to a 10 μg Bafilomycin A1) Bafilomycin A1 were prepared. In control experiments, equal amounts of DMSO (vehicle control) as for the Bafilomycin A1-treated cells were added. The pH probe Lysosensor Blue DND-167 was purchased from ThermoFisher Scientific (Waltham, MA, USA). Nafamostat mesylate was obtained from Sigma-Aldrich (Steinheim, Germany).
9
Cellular Uptake and Cytotoxicity of AFt-2MIM Nanoparticles
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AFt from equine spleen (0.2 μm filtered), 2-methylimidazole (2-MIM), zinc nitrate hexahydrate, thiazolyl blue tetrazolium bromide (MTT), and GOx were purchased from Sigma-Aldrich. DOX was obtained from Tokyo Chemical Industry Company. MDA-MB-231 and MCF-10A cells were obtained from Chinese National Collection of Authenticated Cell Cultures. MX-1 cells were kindly sponsored by Professor Yuqing Chen. LysoSensor Blue DND-167 was obtained from Thermo Fisher Scientific Incorporated. TfR1 (CD71) mouse monoclonal antibody and CoraLite488-conjugated goat anti-mouse lgG(H + L) were purchased from Proteintech Incorporated. Goat anti-Mouse lgG H&L (Alexa FluorR 555) preadsorbed was purchased from Abcam. Blocking buffer, antibody dilution buffer for immunofluorescence, antifade mounting medium with DAPI, and PBS were purchased from Beyotime Biotechnology Incorporated. Fetal bovine serum (FBS), Dulbecco’s modified eagle medium (DMEM), RPMI 1640 medium, penicillin/streptomycin, and 0.25 % trypsin-EDTA solution were purchased from Gibco. BCA protein assay kit was purchased from CoWin Biotech Co., ltd. Amicon Ultra 3 K filters were purchased from Merck Millipore Company. All the chemicals were of analytical grade and used without further purification.
10
Immunofluorescence and live-cell imaging of lysosomes
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Cells grown on 11 mm round glass coverslips were permeabilized, fixed and incubated with antibodies as described previously [29 (link)]. Subsequently, cells were imaged with an LSM 700 confocal microscope (Zeiss, Oberkochen, Germany). For live microscopy cells were seeded into microscopy chambers (8 well μ-slide, Ibidi GmBh, Martinsried, Germany) and incubated with LysoTracker Red DND-99, LysoSensor Blue DND-167, LysoSensor Yellow/Blue DND 160 and CellMask Green (ThermoFisher Scientific, Carlsbad, CA, USA) as described previously [79 (link)]. Intensity of fluorescence and size of lysosomes were determined by ImageJ software (Version 1.5Oi, Bethesda, MD, USA).
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