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BCECF-AM
Ageladine A

Ageladine A is a unique brominated pyridoimidazole alkaloid first isolated from the Japanese marine sponge Agelas nakamuri by Fusetani and co-workers in 2003. Ageladine A has been reported to be a moderate inhibitor of matrix metalloproteases and to be intensely fluorescent.This fluorescence is pH dependent as it is stronger under acidic conditions and barely detectable in alkaline solutions. It is non toxic, highly membrane permeable and is trapped within the cells and acidic organelles through hydrophobic interactions with the inner side of  the membranes. A permeabilisation with acetoxymethyl (AM) esters and subsequent cleavage through cytoplasmic esterases is not involved in this process. So, no toxic by-products of the esterases like formaldehyde and acetic acid arise. In addition, Ageladine A is barely metabolized. This unique features allow long term pH monitoring in cells, tissues and even small transparent whole animals over several days without side effects.


Loading times: Cells in culture around 10-30 minutes.  Small animals (e.g. larves, plathelmintes) need 30-120 minutes. Serum, salt content and ion assembly of the culture medium plays no role. All common buffers and culture media buffered up to pH 7,5 can be used.


Spectral properties: Excitation in a range between 325 nm and 415 nm with maximum at 370 nm.  Emission is between 415 nm and above 500 nm; maximum is at 415 nm. Experiments can be done with common filter settings (like for FURA-2). The fluorescence rises with lower pH in a linear range between pH 4 and pH 8. Quantitative results are achieved with ratiometric methods or FLIM.


Photobleaching occurs, but plays for monitoring at low excitation intensities and exposition times under usual laboratory conditions - even for several days  -  no role.


Leakage was not observed after several days of incubation


Toxic effects were not observed at concentrations up to 30 µM. Patch clamp experiments with PC12 cells showed at concentrations > 10 µM weak changes of the membrane potential.


Recommended concentrations: between 1µM (cells) and 30 µM (whole animals)


Application examples: Fluorescence microscopic monitoring of acidic organelles like lysosomes and endosomes; whole animals like plathelminths, cnidaria; larvae and eggs from different species; screenings; viability tests, Flow cytometry; Fluorescence lifetime imaging microscopy (FLIM)

Pictures


Left: Macrostomum lignano , a Plathelminth. Strong fluorescence intensity of mouth and head region (false color)


Bottom left : Aeolidiella, a mollusk A) picture of whole animal; B) microscopic picture;

C) and D) strong fluorescence   intensity of nematocysts (false color)


Bottom right : PC12-cells with vesicles.


All pictures  with kind permission of the authors

Ageladine A PC12 Zellen Ageladine A Plathelminth Fehlfarben Ageladine A Ganztier

Ageladine A


Intracellular pH-indikator


Properties


- Cell permeabel; loading of cells within 10-30 min

- No esterases involved, therefore no toxic byproducts

- Wide range from pH 4 to pH 8

- Long-term stable; barely metabolized

- For acidic organelles, cells, tissue and small transparent whole animals even in long-term      experiments over several days


The applications as pH indicator (EP2156193) is internationally patented.

The applications for the proof of viability (WO2013186187-A1) is internationally patented.

Downloads

Data sheet Ageladine (pdf)

MSDS Ageladine (pdf)

Literature list (pdf)


LysoGlow84
DMSP

Innovations from the Sea