Lysosome tracer Series probes are a type of fluorescent dye that selectively stain acidic compartments in living cells. These probes have several important characteristics, including:
1) selective labeling of acidic organelles;
2) A nanomolar (nM) concentration can effectively label live cells;
3) Equipped with multi-color probes, multiple standard experiments can be conducted on the sample according to the situation.
Lysosome tracer Structurally composed of a fluorescent group and a connected weak base group, it can freely penetrate the cell membrane and generally aggregate on spherical organelles, suitable for observing the biosynthesis and related pathogenesis inside lysosomes. Lysosome tracer undergoes only partial protonation at neutral pH, so the principle of labeling organelles with this probe may be related to its complete protonation and retention on the organelle membrane. This product Lysosome tracer Green DND-26 is a lysosome probe labeled with green fluorescence, with a maximum excitation/emission wavelength of 504/511nm. This product is provided in the form of a 1mM storage solution dissolved in anhydrous DMSO.
Precautions：
1. Solubility is measured at room temperature, and if the temperature is too low, it may affect its solubility.
2. Our products are non sterile packaging. If used for cell culture, please perform pre-treatment in advance to remove pyrogenic bacteria, otherwise it may cause bacterial contamination.
3. This product is for scientific research purposes only and should not be used for any other purposes.
4. For your safety and health, please wear lab clothes and disposable gloves throughout the operation.
Instructions for Use：
Before use, take out the product and warm it back to room temperature, then perform a brief centrifugation to concentrate the DMSO solution at the bottom of the tube. The optimal working concentration needs to be optimized according to different experimental requirements, cell types, membrane permeability of cells or tissues, etc.
1. Preparation of working fluid
Dilute 1mM storage solution to the working concentration using culture medium or appropriate buffer, with a recommended working solution concentration of 50-75nM;
[Attention]:
1) To reduce the possibility of false positives caused by excessive probe loading, it is recommended to use low concentrations as much as possible without affecting the staining effect.
2) If cells are incubated in a dye free medium after staining, attenuation of fluorescence signals and vacuolization of cells will be observed.
2. Staining
2.1 For adherent cells
1) Place the cells on a cover glass in a culture dish, add suitable culture medium, and allow them to grow on the slide.
2) Once the cells have grown to an appropriate abundance, remove the culture medium and add an appropriate amount of probe containing working solution preheated at 37 ℃. Incubate for 30 minutes to 2 hours in a growing state (specific incubation time depends on cell type).
[Note]: Dynamics studies on the internalization process of Lysosome tracer Green DND-26 indicate that live cells only need a few seconds to uptake this dye.
The disadvantage is that this probe may cause lysosomes to produce an "alkaline effect", which means that prolonged incubation time can induce an increase in lysosome pH. It is recommended that the probe can only be used as a pH indicator when cells are incubated at 37 ℃ for 1-5 minutes.
3) Replace the above staining solution with fresh culture medium and observe under a fluorescence microscope (with appropriate filter). If the dyeing is not sufficient, it is recommended to increase the dye concentration or extend the dyeing time.
2.2 For suspended cells
1) Centrifuge and remove the supernatant.
2) Resuspend cells using probe working solution preheated at 37 ℃ and incubate in a growing state for 30 minutes to 2 hours (specific time depends on cell type).
3) Centrifuge, remove the staining solution, and resuspend the cells in fresh culture medium.
4) Observe under a fluorescent microscope. If the dyeing is not sufficient, it is recommended to increase the dye concentration or prolong the dyeing time.
[Note]: For suspended cells, they can also be attached to cover glass treated with BD Cell Tag, and then stained using a method similar to that of adherent cells.
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