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- Inventor Info
|Antigen/Gene or Protein Targets||Peroxisomes|
S-Glutathionyltrimethyl-2,2’-bipyridyl platinum (IV), PeroxiPlat (Fig. 1) is a new imaging agent based on the luminescent Pt(IV) core2 which rapidly (<5 minutes) and selectively accumulates in peroxisomes and provides high contrast and high resolution imaging of peroxisomes in confocal microscopy (Figure 2). In fluorescence microscopy, PeroxiPlat is best excited at 405nm or 488nm, a standard laser line on many instruments and shows emission between 520-630 nm depending on the exact environment (Figure 3). PeroxiPlat is of low cytotoxicity, showing high percentage survival of cells in an XTT assay at or significantly above the concentrations required for imaging experiments (Figure 4 ).
Peroxisomes are components of almost all human and animal cells which are responsible for a range of vital functions such as breaking down fatty acids, mopping up reactive oxygen molecules such as hydrogen peroxide which are dangerous to other cellular components and the production of certain important phosphilipids used in the brain. Malfunctioning or absence of peroxisomes is associated with a variety of diseases, including Zellwegger Syndrome Spectrum (ZSS) disorders, and peroxisome proliferation is associated with a number of conditions, particularly cancers. However, prior to PeroxiPlat there were no simple small molecule fluorescent agents to allow their study by fluorescence microscopy in live cells, instead methods relied upon expressing fluorescent proteins within the cells.
- Molecular based fluorescence imaging tool
- Rapid (<5m) accumulation and expression in peroxisomes
- Effective in live cells - non disruptive to cell functionality
- High contrast and high resolution in confocal microscopy
- Stable in air and water with a shelf life (dry or frozen) of several months
|Molecular Weight (g/mol)||702|
|In vitro applications||PeroxiPlat selectively accumulates in peroxisomes and provides high contrast and high resolution imaging of peroxisomes in confocal microscopy|
|Solubility||Soluble in DMSO and mixtures of water and DMSO or MeCN (acetonitrile), or in water under basic conditions (pH > 8).|
|Research Area||Cancer, Cell Type or Organelle Marker, Fluorescent Cell Imaging, Structural Biology|
|Chemical Abstracts Service (CAS):||2247040-42-0; 2247040-43-1 (anion and neutral form respectively)|
|Storage||PeroxiPlat for imaging use is best stored in the medium-term as a 5-10 mg/ml solution in either 50% DMSO / water or basic water (pH>8) in a freezer where it is stable for up to a year.|
PeroxiPlat Characterisation, Handling and Storage:
PeroxiPlat is a hygroscopic yellow powder which is indefinitely stable as a dry powder, soluble in DMSO and mixtures of water and MeCN, or in water under basic conditions (pH > 8).
PeroxiPlat may be isolated by evaporation from solutions of organic solvents or by lyophilisation from aqueous solutions. PeroxiPlat solutions should not be acidified below pH 3, and long-term exposure to air leads to oxidation of the glutathione unit and loss of activity.
PeroxiPlat purity may be verified by 1H and 13C NMR (due to the diamagnetic nature of the Pt(IV) ion) and HRMS (ES-ve ion mode) m/z = 701.1728.
PeroxiPlat Example Application Details:
HaCaT cells were grown on glass coverslips in DMEM supplemented with 10% fetal calf serum and 100 U/ml penicillin and 100ug/ml streptomycin at 37oC in a humidified incubator with 5% carbon dioxide. The coverslips were washed briefly in PBS and then incubated in PBS containing PeroxiPlat at 125 μg/ml for 10 minutes at 4oC. The coverslips were then washed in PBS and mounted by inversion on a glass slide. Images were captured on a Zeiss LSM 850 Meta laser scanning confocal microscope. The complex was readily taken up by cells (Figure 2) and showed a punctate staining pattern, characteristic of peroxisomes.
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