Cell Navigator® Lysosome Staining Kit *Deep Red Fluorescence*

Protocol summary

1. Prepare cells
2. Add Lysolite™ Deep Red working solution
3. Incubate at 37°C for 30 - 60 minutes
4. Add Staining Buffer B
5. Incubate at room temperature for 15 - 30 minutes
6. Analyze the cells under fluorescence microscope at Ex/Em = 590/620 nm (Texas Red filter set)

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

Add 10 µL of 500X Lysolite™ Deep Red (Component A) into 5 mL of Live Cell Staining Buffer A (Component B) and mix well to make Lysolite™ Deep Red working solution. Protect from light. Note: 10 µL of 500X Lysolite™ Deep 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

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 1/2 volume (such as 50 µL/well/96-well plate) of Lysolite™ Deep Red working solution.
   
   
3. Incubate the cells in a 37°C, 5% CO₂ incubator for 30 to 60 minutes.
   
   
4. Add 50 µL/well (96-well plate) of Staining Buffer B (Component C) into Lysolite™ Deep Red working solution plate.
   
   
5. Incubate at room temperature for 15 - 30 minutes.
   
   
6. Observe the cells using a fluorescence microscope with Texas Red filter set (Ex/Em = 590/620 nm). Note: The LysoBrite™ Deep Red has minimal or no cell toxicity; it can be used for cell tracking. For cell tracking purpose, skip adding Staining Buffer B: Simply replace the dye-working solution with growth medium, and then observe under fluorescence microscope. 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. Centrifuge the cells at 1000 rpm for 5 minutes to obtain a cell pellet and aspirate the supernatant.
   
   
2. Resuspend the cell pellet gently in pre-warmed growth medium (such as 100 µL/tube), and then add 1/2 volume (50 µL/tube) of Lysolite™ Deep Red working solution.
   
   
3. Incubate the cells in a 37°C, 5% CO₂ incubator for 30 to 60 minutes.
   
   
4. Add 50 µL/tube of Staining Buffer B (Component C) into the Lysolite™ Deep Red working solution plate.
   
   
5. Incubate at room temperature for 15 - 30 minutes.
   
   
6. Observe the cells using a fluorescence microscope with Texas red filter set (Ex/Em = 590/620 nm). Note: The LysoBrite™ Deep Red has minimal or no cell toxicity; it can be used for cell tracking. For cell tracking purpose, skip adding Staining Buffer B: Simply replace the dye-working solution with growth medium, and then observe under fluorescence microscope. 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.