LysoTracker Green dye stains cellular acidic compartments and visualizes enlarged lysosomes. MDA‐MB‐231 cells were stained with LysoTracker Green (Cell Signaling Technology, MA, USA) according to the manufacturer's instructions by incubating them with the dye for 30 min at 37 °C. The fluorescence intensity was measured by flow cytometry (λEx = 488 nm and λEm = 507 nm).
Lysotracker green
Manufactured by Cell Signaling Technology
Sourced in United States
LysoTracker Green is a fluorescent dye that selectively labels acidic organelles, such as lysosomes, in live cells. It is a cell-permeant dye that accumulates in these acidic compartments, allowing for their visualization and study.
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Lab products found in correlation
Lysotracker green, by Thermo Fisher Scientific (1 mentions)
Mitotracker orange, by Thermo Fisher Scientific (1 mentions)
Lysotracker deep red, by Thermo Fisher Scientific (1 mentions)
Ciliobrevin d, by Merck Group (1 mentions)
Mitotracker green, by Cell Signaling Technology (1 mentions)
Ferhonox 1, by Goryo Chemical (1 mentions)
5 protocols using lysotracker green
1
Visualizing Enlarged Lysosomes with LysoTracker
2
Lysosome Staining and Imaging
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LysoTracker Green (Cell signaling technology, U.S.A.) were diluted by 50 μl DMSO and added into normal growth media for a working concentration of 50 nM. After specific treatment, cells were imaged live without fix by 4% paraformaldehyde. The labeled cells were observed at 200× magnifications under a fluorescence microscope.
3
Multimodal Imaging of Mitochondria and Lysosomes
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For the experiments with MitoTracker and LysoTracker labeling, neurons were transfected and labeled with SiR-tz as described above. After washing for 2–3 h, 250 µl of NB Plus + medium containing 100 nM MitoTracker Orange (ThermoFisher Scientific, cat. no. M7510) and 400 nM LysoTracker Green (Cell Signaling Technology, cat. no. 8783 S) was added to the wells that already contained 250 µl of medium, for final concentrations of 50 and 200 nM for MitoTracker and LysoTracker, respectively. For the additional control experiments (data shown in Supplementary Fig. 9 ) involving tetrazine-dye and LysoTracker labeling, MCNs were seeded at a density of 70,000 cells per well and were not transfected. At DIV8, HEPES-diluted TCO*A-Lys was added to cells at a concentration of 250 µM. After 3 days of incubation with TCO*A-Lys, MCNs were labeled with either BODIPY-tz or SiR-tz, washed for 3 h, and then labeled as described above with 200 nM of either LysoTracker Green or LysoTracker Deep Red (ThermoFisher Scientific, cat. no. L12492). Neurons were incubated with MitoTracker and LysoTracker dyes for 30 min and rinsed twice with NB Plus +. Immediately afterward, NB Plus + was replaced by Hibernate E medium (Brain Bits LLC, cat. no. HELF) containing 1% PS and 2% B27 Plus, and neurons were imaged live with confocal scanning microscopy.
4
Culturing NHDFs and Vero Cells for HSV-1 Propagation
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NHDFs and Vero cells were cultured in DMEM containing 5% FBS, 1% l -glutamine, and 1% penicillin-streptomycin (Walsh and Mohr, 2004 (link); Naghavi et al., 2013 (link)). HSV-1 strain F, HSV-1–GFP-Us11 (gifts from I. Mohr, New York University, School of Medicine, New York, NY), and HSV-1 K26GFP (a gift from P. Desai, Johns Hopkins University, Baltimore, MD) were propagated by infecting Vero cells in DMEM containing 1% FBS, 1% l -glutamine, and 1% penicillin-streptomycin (Desai and Person, 1998 (link); Naghavi et al., 2013 (link)). Once 90–100% cytopathic effect was observed, infected cells were scraped into culture medium and lysed by three rounds of −80°C freezing and thawing. Cell debris was removed by centrifugation at 3,000 g for 4 min at 4°C. Stock titers were determined by serial dilution and infection of Vero cells, followed by plaque counting 3 d later. Levels of infectious virus in cultures of NHDFs were determined by freeze-thaw lysis of cells scraped into culture media followed by serial dilution and titration on permissive Vero cells (Walsh and Mohr, 2004 (link)). Ciliobrevin D was procured from EMD Millipore. Phalloidin CF647 was purchased from VWR International. MitoTracker green and LysoTracker green were purchased from Cell Signaling Technology.
5
CRISPR Screening of Neuron Iron and Lysosome Dynamics
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