LysoTracker Blue DND-22 (Synonyms: LysoTracker Blue DND-22; LysoTracker Red DND-99; Lysotracker Green)

Precautions

1) Avoid light during the entire operation.

2) For your safety and health, please wear a lab coat and disposable gloves.

Usage

Before use, take out the product and warm it to room temperature, and centrifuge it at a low speed to make the solution drop to the bottom of the tube. For the first use, please store the product in a single-use aliquot, ≤-20℃ away from light and dry, and try to avoid repeated freezing and thawing. The optimal working concentration needs to be optimized according to different experimental requirements, cell types, and membrane permeability of cells or tissues.

1. Preparation of working solution

Use growth medium or appropriate buffer to dilute the 1mM storage solution to the working concentration. The working solution must be prepared and used immediately. For Lysotracker series lysosomal probes, the recommended working solution concentration is 50-75nM;

【Note】:

1) In order to reduce the false positives that may be caused by excessive probe loading, it is recommended to use as low a concentration as possible without affecting the staining effect.

2) If the cells are incubated in a dye-free medium after staining, the attenuation of the fluorescence signal and the vacuolization of the cells will be observed.

2. Staining steps (adherent cells)

1) Place the cells on a coverslip in a culture dish and add appropriate culture medium to allow them to grow on the slide.

2) When the cells grow to an appropriate density, remove the culture medium and add an appropriate amount of probe-containing culture medium preheated at 37°C (the cells must be completely covered). Incubate the cells for 30 min-2 h under growth conditions suitable for the specific cell type (the specific incubation time depends on the cell type).

3) Replace the above staining solution with fresh culture medium without probes and observe with a fluorescence microscope equipped with a suitable filter (see Appendix 1). If the staining is not sufficient, it is recommended to increase the dye concentration or extend the staining time so that the dye can accumulate on the lysosomes.

3. Staining steps (suspended cells)

1) Centrifuge the sedimented cells and remove the supernatant.

2) Resuspend the cells with an appropriate amount of probe-containing culture medium preheated at 37°C and incubate the cells for 30 min-2 h under growth conditions suitable for the specific cell type (the specific incubation time depends on the cell type).

3) Centrifuge, remove the staining solution, and add fresh culture medium to resuspend the cells.

4) Observe with a fluorescence microscope equipped with appropriate filters. If the staining is not sufficient, it is recommended to increase the dye concentration or extend the staining time so that the dye can accumulate on the lysosomes.