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LysoPrime Green – High Specificity and pH Resistance

Item # Unit Size
L261-10
10 µL x 1
L261-12
10 µL x 3

For Research Use Only Products

Content: 10 μL x 1; 10 μL x 3

∼ Features ∼
– High selectivity for lysosomes
– High retention even in lysosomes that had pH neutralized after staining
– Long-term visualization of lysosomes for live cell imaging

The general number of usable assays per 10 μL:
・35 mm dish x 10
・96 well plate x 2

Storage Condition: Store at -80oC
Shipping Condition: with blue ice




Detection Principle



Lysosomes are involved in a multitude of cellular processes, and their dysfunction is associated with various diseases. Understanding lysosome-related physiology requires lysosome-specific probes that can track the biological processes of lysosomes within cells. Compared to other subcellular components, lysosomes are the most acidic organelles. Based on this feature, most lysosomal probes have been designed to target the acidic environment within lysosomes.

However, the current strategies for monitoring lysosome activities exhibit several limitations. As consequence of being designed simply to target acidic environments, most lysosomal probes have poor specificity towards the lysosome and stain many non-lysosomal acidic compartments. Moreover, existing lysosome probes are highly reliant on the lysosomal pH value; their emission intensity is unstable and varies significantly due to differences in acidity among lysosomes within a cell.

We have developed a novel fluorescent probe, LysoPrime Green, in order to track the lysosome while avoiding these aforementioned restrictions. Compared to existing lysosome probes, LysoPrime Green shows better selectivity towards lysosomes, and its fluorescence is not affected by variations in lysosome acidity that can occur after staining. In addition, the high-retentivity of LysoPrime Green enables long-term imaging
experiments.

LysoPrime Green’s High Selectivity Towards Lysosomes



LysoPrime Green co-localized with LAMP-1 to a higher degree than existing reagent.

 
LysoPrime Green’s Lysosomal pH Independence



Control: Normal condition, Bafilomycin A1: Inhibition of lysosomal acidification

LysoPrime Green retained lysosome staining even in cells that had lysosomal pH neutralized by Baf A1 treatment.

 
Observation of the lysosomal pH change in HeLa cells



Control: Normal condition, Bafilomycin A1: Inhibition of lysosomal acidification
LysoPrime Green filter sets: 488 nm (Ex), 500 – 570 nm (Em)
Reagent LTR (Company T) filter sets: 548 nm (Ex), 550 – 650 nm (Em)

<Microplate reader>



 

Fluorescence Properties



λex : 456 nm
λem : 540 nm

<Recommended filter settings>
Ex : 400 - 490 nm, Em : 500 - 600 nm
Co-Staining with Autophagy Dye and LysoPrime Green under Autophagy-Inducing Conditions



LysoPrime Green retained Lysosome staining after 2 h of starvation, while Reagent LTG (Company T) lost lysosome localization.

*DAPRed (D677): Autophagy detection dye designed for monitoring the autophagosome.

 
Co-Staining with Mitophagy Dye and LysoPrime Green under Mitophagy-Inducing Conditions



LysoPrime Green retained lysosome staining after 24 h FCCP treatment, while Reagent LTG (Company T) lost lysosome localization.

* Mtphagy Dye (MD01): Mitophagy Detection dye designed for monitoring mitophagy.
Is this right that the transportation temperature and storage temperature (-80°C) are different?

No problem. There is almost no deterioration during transportation at room temperature. The storage condition is set at -80°C because the purity of the lysosomes was degraded when stored at temperatures other than -80°C for more than one month. We have confirmed that storage at room temperature (25°C) for one month does not affect the staining performance of lysosomes.




Is seems like that after staining some of the organelles has been stain besides lysosome is stained.

We have confirmed that the presence of excessive amounts of dye causes staining of nuclei and cytoplasm in addition to lysosomes. The optimal staining concentration is also affected by cell type and density, so be sure to check the optimal concentration before using the product for the first time. We have confirmed that working solution diluted 2,000-4,000 times with HBSS for 30 minutes can stain lysosomes without any problem.


*Scale bar: 10 µm




Is it possible to stain with serum-containing medium?

Since LysoPrime Green is affected by serum, be sure to prepare working solution in serum-free solution such as HBSS or serum-free medium.




Should I use drug stimulation before or after staining?

LysoPrime Green accumulates in lysosomes in a pH-dependent manner and remains in the lysosomes even if the pH is changed after accumulation. On the other hand, LysoPrime Green cannot accumulate in lysosomes whose pH is changed to neutral by drug stimulation. Therefore, we recommend drug stimulation after staining to minimize the effect on staining ability.