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1. Prepare Lysosome-staining solution:

1.1    Warm LysoBrite™ dyes to room temperature.

1.2    Prepare dye working solution by diluting 20 µL of 500 X LysoBrite™ dyes to 10 mL of Hanks and 20 mm Hepes buffer or buffer (HBSS) of your choice.

Note 1: 20 µL of LysoBrite™ dye is enough for one 96-well plate. Aliquot and store unused LysoBrite™ dye stock solutions at < -15 ^oC. Protect it from light and avoid repeated freeze-thaw cycles.

Note 2: 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.

2. Prepare and stain cells:

2.1    For adherent cells: a). 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 the dye-working solution (from Step 1.2). b). Incubate the cells in a 37 °C, 5% CO₂ incubator for 30 minutes. c). 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. d). Observe the cells using a fluorescence microscope fitted with a desired 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.

2.2    For suspension cells: a). Add equal volume of dye-working solution (from Step 1.2) into the cells. Incubate the cells in a 37 °C, 5% CO₂ incubator for 30 minutes. b). 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. c). Observe the cells using a fluorescence microscope equipped with a desired filter set.

Note 1: 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 2: Suspension cells may be attached to cover-slips that have been treated with BD Cell-Tak^® (BD Biosciences) and stained as adherent cells (see Step 2.1).

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