Cell Navigator® Lysosome Staining Kit *Orange Fluorescence*

Protocol summary

1. Prepare cells
2. Add LysoBrite™ Orange working solution
3. Incubate at 37°C for 30 minutes
4. Wash the cells
5. Analyze the cells under fluorescence microscope at Ex/Em = 540/570 nm (TRITC filter set)

Important notes
Thaw all the kit components at room temperature before starting the experiment.

20 µL of 500X LysoBrite™ Orange (Component A) to 10 mL of Live Cell Staining Buffer (Component B) to make LysoBrite™ Orange working solution. Note: 20 µL of 500X LysoBrite™ Orange (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

For adherent cells:

1. 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.
   
   
2. When cells reach the desired confluence, add equal volume of LysoBrite™ Orange working solution.
   
   
3. Incubate the cells in a 37°C, 5% CO₂ incubator for 30 minutes.
   
   
4. 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.
   
   
5. Observe the cells using a fluorescence microscope with TRITC filter set (Ex/Em = 540/570 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:

1. Add equal volume of LysoBrite™ Orange working solution into the cells.
   
   
2. Incubate the cells in a 37°C, 5% CO₂ incubator for 30 minutes.
   
   
3. 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.
   
   
4. Observe the cells using a fluorescence microscope with TRITC filter set (Ex/Em = 540/570 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.