Cells were fixed in 4% paraformaldehyde, permeabilised and incubated with allophycocyanin-labelled anti-IL23R (FAB14001P, R&D Systems), Rab11, Rab7 (Cell Signaling), or DAPI (Thermo Fisher) and Cy2-labelled secondary antibodies. For LysoTracker (Cell Signaling) cells were stained prior to fixation. Fluorescence microscopy used the Zeiss Axio Observer microscope (Carl Zeiss Microscopy, Thornwood, NY). Colocalisation was quantified using ImageJ.
Lysotracker
Manufactured by Cell Signaling Technology
Sourced in France, United States
LysoTracker is a fluorescent dye that selectively stains acidic organelles, such as lysosomes, within living cells. It enables the visualization and tracking of lysosomal dynamics in real-time.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Rab11, by Cell Signaling Technology (1 mentions)
Fab14001p, by R&D Systems (1 mentions)
Er tracker, by Cell Signaling Technology (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Poly adp ribose polymerase parp antibody, by Cell Signaling Technology (1 mentions)
Caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Mitotracker, by Thermo Fisher Scientific (1 mentions)
Er tracker, by Thermo Fisher Scientific (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Rabbit anti mouse igg igm h l hrp secondary antibody, by Thermo Fisher Scientific (1 mentions)
Axioskop 2 plus fluorescence microscope, by Zeiss (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Mitotracker, by Cell Signaling Technology (1 mentions)
7 protocols using lysotracker
1
Immunofluorescence Analysis of IL23R
2
Lysosome Evaluation in MM Cells
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MM cells were exposed to AMG9810 (5–10 µM), bortezomib (3–5 nM) or their combination for 24 or 48 h, labeled with LysoTracker (Cell Signaling Technology) for 30 min, 37 °C for 30 min, and analyzed by flow cytometry.
3
Mitochondria, Lysosomes, and ER Intercellular Transfer
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To determine whether the TNTs allow the transport of mitochondria, transfected cells were divided in two groups. Each group were labeled separately by either Green or Red MitoTracker dyes (Molecular Probes) for 30 min and then washed extensively with PBS before mixing the two populations in the same culture dish. After 1 h of incubation, the cells were either fixed or subjected to the time-lapse imaging. We quantified the number of cells that contain both Mitotracker (yellow color).
To investigate the transfer of either lysosome or endoplasmic reticulum between the cells, Lyso Tracker™ or ER tracker ™ (cell signaling) were added directly into normal growth media for a working concentration of 50 nM or 2 μM respectively according to the manufactures structures and then analyzed immediately by real-time acquisition.
To investigate the transfer of either lysosome or endoplasmic reticulum between the cells, Lyso Tracker™ or ER tracker ™ (cell signaling) were added directly into normal growth media for a working concentration of 50 nM or 2 μM respectively according to the manufactures structures and then analyzed immediately by real-time acquisition.
4
Cell Viability, Apoptosis, and Oxidative Stress Assays
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RPMI 1640 medium, RPMI red-phenol-free medium, fetal bovine serum (FBS), L-glutamine, and penicillin–streptomycin were purchased from Gibco BRL (Cergy-Pontoise, France). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), human anti-β-actin antibody, cell death detection enzyme-linked immunosorbent assayPLUS (ELISA), and 2′,7′-dichlorofluorescéine diacetate (DCFDA) were obtained from Sigma-Aldrich (Saint-Quentin-Fallavier, France). LysoTracker, goat anti-rabbit IgG H&L horseradish peroxidase (HRP) secondary antibody, Poly-ADP-ribose polymerase (PARP) antibody, caspase-3 antibody, and cleaved caspase-3 antibody were purchased from Cell Signaling Technology—Ozyme (Saint-Quentin-en-Yvelines, France). MitoTracker, ER-Tracker, and rabbit anti-mouse IgG-IgM H&L HRP secondary antibody were obtained from Invitrogen—Thermo Fisher Scientific (Villebon-sur-Yvette, France). Immobilon Western Chemiluminescent HRP Substrate was acquired from Merck (Lyon, France).
5
Lysosomal Biogenesis in Macrophages
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Assessment of the lysosomal biogenesis in macrophages was performed as described previously.38 After treatment, cells were washed three times with PBS, followed by incubation with LysoTracker (Cell Signaling Technology) for 30 min. The intralysosomal pH in macrophages was estimated using LysoSensor (Dalian Meilun Biotechnology) according to the manufacturer's process. Briefly, cells were washed three times with PBS, followed by incubation with LysoSensor for 30 min. The fluorescence was visualized using a Zeiss Axioskop 2 plus fluorescence microscope (Carl Zeiss). The fluorescence intensity was analysed using ImageJ software (NIH).
6
Tracking PB Aptamer Lysosomal Escape
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To test whether PB aptamer can escape from lysosome after cellular internalization, HUVEC was cultured in chamber slides and incubated with 100 nM PB aptamer for 1 hour, 4 hours, and 6 hours, respectively. Lysotracker (1:100 diluted, Cell Signaling) was added 0.5 hour prior to the end. After the incubation, medium was removed and cells were washed by PBS, followed by nuclear staining with DAPI. Cells were observed under a Nikon Eclipse 80i microscope.
7
Mitophagy Evaluation in HK-2 Cells
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LysoTracker and MitoTracker were obtained from Cell Signaling Technology (Beverly, USA) to assess the level of mitophagy. HK-2 cells were cultured in a six-well plate and treated with specific drugs. After incubation according to the manufacturer's protocol, the plate was detected by microscopy, and images were captured in random fields. Red fluorescence represents mitochondria, and green fluorescence represents lysosomes.
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