SKU: L261 Categories: ,

LysoPrime Green – High Specificity and pH Resistance

$120.00$250.00

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


*This offer is for the trial size kit of LysoPrime Green (L261-10).


LDs/Mitochondria/Senescence Map Selection Guide

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DescriptionDataQ & AManualS.D.S

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*This offer is for the trial size of LysoPrime Green (L261-10).

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<Storage Precautions>
・For long-term storage, storage below -80°C or in liquid nitrogen is recommended.
・Even store under -20°C conditions may cause degradation over time.
If storing below -80°C is difficult, please consider using the reagent as soon as possible.
・Please avoid repeated freezing and thawing, as it may cause degradation.


Detection Principle

Lysoprime Green enters the acidic lysosome environment and remains in the lysosome, even after pH has been neutralized (due to lysosomal dysfunction, etc.). Conventional lysosome dyes such as LysoView and LysoTracker will diffuse out of the lysosome when lysosome pH has been neutralized.

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

Two co-localization experiments between a LysoTracker Green and LAMP1. The first is a co-stain of LysoPrime Green and LAMP1. The second is a co-stain of a conventional lysosome dye and LAMP1

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

 

LysoPrime Green’s Lysosomal pH Independence

LysoPrime Green and LysoTracker Green being compared under control conditions and under Bafilomycin A1 treatment. LysoPrime Green signal is consistent, whereas LTG signal is greatly diminished under Bafilomycin A1.

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

<Experimental Conditions>
Control: Normal condition, Bafilomycin A1: Inhibition of lysosomal acidification
Green: Ex= 488 nm, Em= 500-570 nm
Scale bar: 20 µm

 

High retention in lysosome

The lysosomal retention of LysoPrime Green and existing dyes were compared using cells stained with each dye. While the fluorescence of the existing dye decreased 90 minutes after staining, LysoPrime Green maintained its fluorescence intensity and showed high retention.

LysoPrime Green and LysoTracker Green being compared at different times after staining. LysoTracker Green sees a considerable decrease in intensity.

<Experimental Conditions>
Green: Ex= 488 nm, Em= 500-570 nm
Scale bar: 10 µm

 

In addition, HeLa cells stained with LysoPrime Green or the existing product were trypsinized and recovered, and the fluorescence intensity was checked using a flow cytometer. In contrast, LysoPrime Green showed little difference in fluorescence intensity between pre-and post-trypsin treatment staining. LysoPrime Green was found to have low leakage of dye due to pretreatment.

The procedures for trypsinization and staining are described. Flow cytometer measurements of fluorescence intensity are much more similar for the LysoPrime Green dye than the LysoTracker Green, between the groups stained before trypsinization and the groups stained after trypsinization.

 

 

 Evaluation using lysosomal acidity inhibitors (co-staining with LysoTracker™ Red)

LysoPrime Green and LysoTraker™ Red were co-stained with LysoPrime Green and LysoTraker™ Red, respectively, and the lysosomal inhibitor Bafilomycin A1 was added for fluorescence imaging. The fluorescence signal of LysoPrime Green was almost unchanged with or without Bafilomycin A1, whereas LysoTraker™ Red showed a decrease in fluorescence signal due to lysosomal acidification caused by the addition of Bafilomycin A1. Thus, the combination of both reagents allows simultaneous evaluation of lysosome quantity and pH change. Furthermore, this fluorescence intensity could be quantified using a plate reader.

Graphs indicate the loss of LysoTracker Red intensity in presence of Bafilomycin A1.

 

<Experimental Conditions>
Control: Normal condition, Bafilomycin A1: Inhibition of lysosomal acidification
LysoPrime Green filter sets: 488 nm (Ex), 500 – 570 nm (Em)
LysoTracker Red filter sets: 548 nm (Ex), 550 – 650 nm (Em)


Fluorescence Properties

LysoPrime Green's excitation and emission spectra

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

2 experiments: One is a co-stain between LysoPrime Green and DAPRed. The other is a co-stain between LysoTracker Green and DAPRed. DAPRed Autophagy signal increases after 2 hours. LysoPrime Green is seen co-localizing with DAPRed after 2 hours. LysoTracker Green has its signal significantly diminished after 2 hours and co-localizes with DAPRed to a much smaller degree

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

All co-stain results are from 24 hours after staining. LysoPrime Green is largely retained and co-localizes with Mtphagy dye under FCCP treatment. LysoTracker Green is largely diminished under control conditions and does not co-localize as well with Mtphagy under FCCP treatment.

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.

 

Exosome localization in intracellular organelles

Co-stain experiments between LysoPrime Green and ExoSparkler Deep Red reveal co-localization of the two dyes

LysoPrime Green retained lysosome staining after 240 min, time-dependent exosome localization in lysosome was observed.

*ExoSparkler Deep Red (EX03) :ExoSparkler designed for exosome membrane labeling.

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.
Can I use LysoPrime Green with fixed cells?
No, LysoPrime Green is suitable for live cells.
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.

Results indicate that excessive amounts of dye will cause staining of nuclei and cytoplasm in addition to lysosomes. Diluting the working solution 2000 to 4000 times with HBSS and applying for 30 min will stain lysosomes without any problems

*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.

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