Cell Navigator® Lysosome Staining Kit *Red Fluorescence*
![Images of HeLa cells stained with A: Cell Navigator® Lysosome Staining Kit (Cat# 22658), B: LysoTracker® Red DND-99 (from Invitrogen) in a Costar black wall/clear bottom 96-well plate. The signals were compared at 0 and 120 seconds exposure time by using an Olympus fluorescence microscope.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-navigator-lysosome-staining-kit-red-fluorescence%2Ffigure-for-cell-navigator-lysosome-staining-kit-red-fluorescence_FSspV.jpg&w=640&q=75)
![Images of HeLa cells stained with A: Cell Navigator® Lysosome Staining Kit (Cat# 22658), B: LysoTracker® Red DND-99 (from Invitrogen) in a Costar black wall/clear bottom 96-well plate. The signals were compared at 0 and 120 seconds exposure time by using an Olympus fluorescence microscope.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-navigator-lysosome-staining-kit-red-fluorescence%2Ffigure-for-cell-navigator-lysosome-staining-kit-red-fluorescence_FSspV.jpg&w=640&q=75)
![Images of HeLa cells stained with A: Cell Navigator® Lysosome Staining Kit (Cat# 22658), B: LysoTracker® Red DND-99 (from Invitrogen) in a Costar black wall/clear bottom 96-well plate. The signals were compared at 0 and 120 seconds exposure time by using an Olympus fluorescence microscope.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-navigator-lysosome-staining-kit-red-fluorescence%2Ffigure-for-cell-navigator-lysosome-staining-kit-red-fluorescence_FSspV.jpg&w=128&q=25)
![Image of Hela cells stained with Cell Navigator® Lysosome Staining Kit in a Costar black wall-clear bottom 96-well plate.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-navigator-lysosome-staining-kit-red-fluorescence%2Ffigure-for-cell-navigator-lysosome-staining-kit-red-fluorescence_gbra0.jpg&w=128&q=25)
![Live PDAC microtumours feed on dead-cell debris. (a,b) Time-lapse images of live and dead cell assays. A live PCI-55 microtumour showed that an early apoptotic cell underwent late apoptosis on the surface of microtumours, and was then taken into their bodies (a). PCI-55 microtumours that took up Annexin V<sup>+</sup>/EthD-1<sup>+</sup> cells massively accumulated Annexin V on their surfaces, whereas EthD-1 was dispersed throughout their bodies (b). (c–i) Feeding dead-cell debris to anchorage-dependent PCI-55 microtumours. To maintain PCI-55 microtumours anchored to the micro/nanoplate, we added their UV-induced dead-cell debris. Schematic of the protocol (c). Time-lapse images (d). Time after adding dead-cell debris shown as hh:mm. Grown microtumours devoured dead-cell debris aggressively (e). Tumour sizes in PCI-55 microtumours at 48 h after dead-cell feeding (f–h), CFSE-labelled PCI-55 microtumours were fed UV-induced dead PCI-55 cells into which Edu (red fluorescence) had been incorporated. 3D images of CFSE-labelled PCI-55 microtumours with added Edu<sup>+</sup> dead-cell debris (g). Confocal images of CFSE-labelled PCI-55 microtumours with added Edu<sup>+</sup> dead-cell debris. Cross sections of CFSE-labelled PCI-55 microtumours taking up debris-derived Edu (h). Fluorescence for indicated markers of PCI-55 microtumours taking up debris-derived Edu (i). Zoomed-in image shows that Edu<sup>+</sup> dead-cell debris was endocytosed from the surface of the PDAC microtumour and then incorporated into lysosomes in the inner cells forming the microtumours. Source: <strong>Visualising the dynamics of live pancreatic microtumours self-organised through cell-in-cell invasion</strong> by Miyatake et al., <em>Scientific Reports</em>, Sept. 2018.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-navigator-lysosome-staining-kit-red-fluorescence%2Ffigure-for-cell-navigator-lysosome-staining-kit-red-fluorescence_gyq2i.jpg&w=128&q=25)
![Cell Navigator Lysosomal Stain](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-navigator-lysosome-staining-kit-red-fluorescence%2Ffigure-for-cell-navigator-lysosome-staining-kit-red-fluorescence_QNmJI.jpg&w=128&q=25)
![Lysosome localization and motility is altered for starvation-induced Hela cells. A: Healthy untreated Hela cells. Lysosomes (Red) were dispersed widely throughout the cytosol in cells. B: Starved Hela Cells. The cells were starved for 24 hours (no serum), and lysosomes were aggregated in the perinuclear region. Nuclei were stained with Hoechst 33342.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-navigator-lysosome-staining-kit-red-fluorescence%2Ffigure-for-cell-navigator-lysosome-staining-kit-red-fluorescence_drqK6.jpg&w=128&q=25)
AT A GLANCE
Protocol summary
- Prepare cells
- Add LysoBrite™ Red working solution
- Incubate at 37°C for 30 minutes
- Wash the cells
- Analyze the cells under fluorescence microscope at Ex/Em = 575/600 nm (TRITC filter set)
Important notes
Thaw all the kit components at room temperature before starting the experiment.
PREPARATION OF WORKING SOLUTION
Add 20 µL of 500X LysoBrite™ Red (Component A) to 10 mL of Live Cell Staining Buffer (Component B) to make LysoBrite™ Red working solution. Protect from light. Note: 20 µL of 500X LysoBrite™ Red (Component A) is enough for one 96-well plate. The optimal concentration of the fluorescent lysosome indicator varies depending on the specific application. The staining conditions may be modified according to the particular cell type and the permeability of the cells or tissues to the probe.
For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
SAMPLE EXPERIMENTAL PROTOCOL
For adherent cells:
- Grow cells either in a 96-well black wall/clear bottom plate (100 µL/well/96-well plate) or on cover-slips inside a petri dish filled with the appropriate culture medium.
- When cells reach the desired confluence, add equal volume of LysoBrite™ Red working solution.
- Incubate the cells in a 37°C, 5% CO2 incubator for 30 minutes.
- Wash the cells twice with pre-warmed (37°C) Hanks and 20 mM Hepes buffer (HBSS) or buffer of your choice, fill the cell wells with HBSS or growth medium.
- Observe the cells using a fluorescence microscope with TRITC filter set (Ex/Em = 575/600 nm). Note: It is recommended to increase either the labeling concentration or the incubation time to allow the dye to accumulate if the cells do not appear to be sufficiently stained.
For suspension cells:
- Add equal volume of LysoBrite™ Red working solution into the cells.
- Incubate the cells in a 37°C, 5% CO2 incubator for 30 minutes.
- Wash the cells twice with pre-warmed (37°C) Hanks and 20 mM Hepes buffer (HBSS) or buffer of your choice, fill the cell wells with HBSS or growth medium.
- Observe the cells using a fluorescence microscope with TRITC filter set (Ex/Em = 575/600 nm). Note: It is recommended to increase either the labeling concentration or the incubation time to allow the dye to accumulate if the cells do not appear to be sufficiently stained. Suspension cells may be attached to cover-slips that have been treated with BD Cell-Tak® (BD Biosciences) and stained as adherent cells.
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