All uptake and colocalization experiments were carried out with the HF488-labeled Aβ peptides using a Leica SP5 confocal microscope (Leica Microsystems). For colocalization with FM 4–64 (Life Technologies), 5 μg/ml of the dye was added together with the peptide, and images were recorded after 1 hr of incubation. Both dyes were excited by the 488-nm laser line. HF488 emission was collected from 520–580 nm and FM 4–64 emission between 700–800 nm. For colocalization with LysoTracker red (Life Technologies), cells were incubated with HF488-labeled peptide for 24 hr, washed once with serum-free medium, and thereafter incubated for 1 hr with 100 nM LysoTracker red before images were recorded. LysoTracker red was excited using the 647-nm laser line, and emission was collected between 700–800 nm. Sequential acquisition was used in all experiment to avoid bleed-through and cross-excitation. Cellular uptake at 4°C was assessed by keeping the cells in a cold room during incubation. The cells were washed prior to imaging to avoid uptake while observing the cells on the microscope.
Lysotracker red
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Germany, Italy, United Kingdom
LysoTracker Red is a fluorescent dye that selectively stains acidic organelles, such as lysosomes, in live cells. It is a useful tool for visualizing and tracking the dynamics of these organelles within the cellular environment.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (43 mentions)
Mitotracker green, by Thermo Fisher Scientific (40 mentions)
Mitotracker red, by Thermo Fisher Scientific (27 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (18 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (17 mentions)
Lsm 710, by Zeiss (16 mentions)
Lsm 780 confocal microscope, by Zeiss (10 mentions)
Chloroquine, by Merck Group (9 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (9 mentions)
Lsm 510, by Zeiss (9 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (9 mentions)
Sp5 confocal microscope, by Leica (8 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (8 mentions)
Lsm 700, by Zeiss (8 mentions)
Lsm 880, by Zeiss (8 mentions)
Lysotracker green, by Thermo Fisher Scientific (8 mentions)
Er tracker red, by Thermo Fisher Scientific (7 mentions)
Yoyo 1, by Thermo Fisher Scientific (7 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (7 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
467 protocols using lysotracker red
1
Cellular Uptake and Colocalization of Aβ Peptides
2
Immunofluorescence Analysis of Autophagic Markers
3
Imaging Mitochondrial Dynamics and Lysosome Motility
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Cells were imaged for 8 hours, starting 24 hours after transfection, for both siRNA and overexpression experiments. Cells already displaying mitochondrial fragmentation at the beginning of the imaging period were excluded from analysis. Live cell imaging was performed using a Nikon A1R confocal microscope, enclosed in a heating chamber at 37°C with 5% CO2 perfused over the plate holder. Cells were plated on 35 mm live-cell imaging dishes and imaged using a 63X (NA 1.4) objective. Time-lapse images were taken every 20 seconds over a period of 8 hours or as indicated. For experiments using only GFP-labeled proteins, the fluorophore was excited using the 488-nm line of an Ar ion laser. For time-lapse sequences where dual labels were used (Lysotracker red; Molecular Probes, OR; and GFP-Mito), the fluorophores were sequentially excited with the 488 and 543 laser lines and emission signals were separated using an electronically controlled emission discrimination filter with the appropriate filter sets. To monitor lysosome movements, Lysotracker red (Molecular Probes) was added to cells 30 minutes prior to imaging. To visualize early or late endosome motility we expressed GFP-RAB5 or GFP-RAB7, respectively.
4
Amyloid-β Internalization and Organelle Colocalization
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About 5 × 104 cells/ml were grown on 25 mm round glass coverslips (Carl Roth GmbH, Karlsruhe, Germany) and incubated for 3, 8, and 18 h with 400 nM FITC-labelled Aβ1–42 peptide. Imaging was performed in PBS containing 5% FBS at room temperature using the Leica confocal system TCS SP5 II with the software LAS AF (version 2.60) (Leica Microsystems CMS GmbH, Heidelberg, Germany). Incubation with ER-Tracker™ Red (λEX/λEM = 587/615 nm), MitoTracker® Red CM-H2Xros (λEX/λEM = 579/599 nm), or LysoTracker® Red (λEX/λEM = 577/590 nm) (Molecular Probes, Invitrogen, Eugene, OR) was performed 15 or 5 min (for LysoTracker Red) prior to imaging. FITC and ER-Tracker/MitoTracker/LysoTracker Red were sequentially excited with an argon laser at 488 nm and with a yellow diode at 561 nm, respectively. Images (512 × 512 pixels) were acquired by sequential scanning between lines (line average 6) using 40x (1.3 NA) oil-immersion objective with a 12-bit HyD detector at corresponding spectral range for each fluorophore. Images were overlayed in ImageJ software (version 1.48) (http://imagej.nih.gov/ij/ ) for putative detection of fluorescence signal colocalization.
5
Visualizing Endosomal Acidification
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Endosomal acidification was assessed with LysoTracker Red (Molecular Probes) as a probe for low-pH organelles. Cells were pretreated with DMSO or inhibitors for 2 h at 37 °C and then incubated with 75 nM LysoTracker Red (Molecular Probes) for an additional 1 h. Cells were fixed with 4% paraformaldehyde and analyzed by confocal fluorescence microscopy. Also, HCL (10 μM) and BAF (200 nM) were used as controls when indicated.
6
Lysosomal Activity Assay Protocol
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The alteration of LMP was assayed as previously described [30 (link)]. For detection of the acidic cellular, LysoTracker Red (Invitrogen, USA) was used, which emits bright red fluorescence in acidic vesicles, after treatment of the cells with the agent, LysoTracker Red (the concentration used as recommended), for a period of 30 min. Following PBS washing, the fluorescent micrographs were captured using an inverted fluorescence microscope (Nikon eclipse Ts2, Japan).
7
Lysosomal Staining Protocol
8
Assessing Lysosomal Acidification via LysoTracker
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Lysosome acidification was determined using LysoTracker Red (Invitrogen, L7528) as previously described.21 Primary TECs isolated from mice and HK‐2 cells were harvested as described above and incubated with 50 nmol/L LysoTracker Red for 30 min at 37°C in the dark. The mean fluorescent intensity of LysoTracker Red was quantified by flow cytometry.
9
Fluorescence Imaging of Lysosomal Localization
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The COS-7 cells (1 × 106 cells mL–1) were seeded in confocal dish. After being cultured for 24 h, cells were added with 10 µM of DAMB-SA, DAMB-SAN in culture medium and incubated for 30 min at 37 °C. Then, cells were washed by PBS buffer (pH = 7.4) and imaged by using a confocal laser scanning microscope (CLSM) (LSM880, Carl Zeiss, Germany). Capture condition: λex = 405 nm, λem = 540–600 nm.
The COS-7 cells were added with 10 µM of DAMB-SA in culture medium and incubated for 15 min at 37 °C, then added with 1 µM of BODIPY (Invitrogen, USA) and Lyso-Tracker Red (Invitrogen, USA) in culture medium and incubated for 15 min. Then, cells were washed by PBS buffer and imaged by CLSM. The COS-7 cells were added with 10 µM of DAMB-SAN in culture medium and incubated for 15 min at 37 °C, then added with 1 µM of Lyso-Tracker Red (Invitrogen, USA) in culture medium and incubated for 15 min. Then, cells were washed by PBS buffer and imaged by CLSM (LSM880, Carl Zeiss, Germany). Capture condition: BODIPY: λex = 488 nm, λem = 500–530 nm; Lyso-Tracker Red: λex: 543 nm, λem: 580–650 nm.
The COS-7 cells were added with 10 µM of DAMB-SA in culture medium and incubated for 15 min at 37 °C, then added with 1 µM of BODIPY (Invitrogen, USA) and Lyso-Tracker Red (Invitrogen, USA) in culture medium and incubated for 15 min. Then, cells were washed by PBS buffer and imaged by CLSM. The COS-7 cells were added with 10 µM of DAMB-SAN in culture medium and incubated for 15 min at 37 °C, then added with 1 µM of Lyso-Tracker Red (Invitrogen, USA) in culture medium and incubated for 15 min. Then, cells were washed by PBS buffer and imaged by CLSM (LSM880, Carl Zeiss, Germany). Capture condition: BODIPY: λex = 488 nm, λem = 500–530 nm; Lyso-Tracker Red: λex: 543 nm, λem: 580–650 nm.
10
Lysosomes Imaging and Quantification
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Acidic vesicles were labeled with LysoTracker Red (L7528; Thermo Fisher Scientific). Cells were seeded on glass coverslips and grown in complete medium (DMEM, 10% serum) for 24 h before adding LysoTracker Red (Thermo Fisher Scientific) directly in the medium (final concentration: 75 nM) and Hoechst 33342 for nuclear staining. After 30 min at 37°C, the cells were washed once with PBS and fixed for 15 min with 4% paraformaldehyde in PBS. After two washes with PBS, coverslips were mounted on a glass slide with Vectashield (H-1200; Vector Laboratories). The dells were imaged with an Olympus BX-51 fluorescence microscope (60× magnification), and images were processed with ImageJ for quantification. Briefly, color channels were split, and the red channel images were processed with “Find edges” before threshold adjustment and particle size determination. Statistical differences (t test) were determined with Prism 5 (GraphPad Software Inc.).
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