Cell Navigator® Lysosome Staining Kit *Blue Fluorescence*

Protocol summary

1. Prepare cells
2. Add dye working solution
3. Incubate at 37°C for 30 minutes to 2 hours
4. Analyze under fluorescence microscope at Ex/Em = 360/445 nm (DAPI filter set)

Important notes
Warm LysoBrite™ Blue (Component A) to room temperature.

Dilute 20 µL of LysoBrite™ Blue (Component A) into 10 mL of Live Cell Staining Buffer (Component B). Protect from light. Note: 20 µL of LysoBrite™ Blue (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

1. Prepare samples and cells.
   
   For adherent cells: Grow cells either in a black wall/clear bottom 96-well plate (100 µL/well/96-well plate) or on coverslips inside a petri dish filled with the appropriate culture medium. When cells reach the desired confluence, add equal volume (such as 100 µL/well/96-well plate) of the dye-working solution.
   
   For suspension cells: Centrifuge the cells at 1,000 rpm for 5 minutes to obtain a cell pellet and aspirate the supernatant. Resuspend the cell pellet gently in pre-warmed growth medium, and then add equal volume of the dye-working solution.
   
   
2. Incubate the cells in a 37°C, 5% CO₂ incubator for 30 minutes to 2 hours.
   
   
3. Observe the cells using a fluorescence microscope fitted with a DAPI filter set. 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. Note: Suspension cells may be attached to coverslips that have been treated with BD Cell-Tak^® (BD Biosciences) and stained as adherent cells.