Si-DMA for Mitochondrial Singlet Oxygen ImagingMT05 Product Manual

Singlet oxygen (¹O₂) is one of the Reactive Oxygen Species (ROS). ¹O₂ is known to be a cause of spots and wrinkles of the skin due to its very strong oxidizing potential. In the field of cancer research, ¹O₂ is of a particular importance because of its key role in photodynamic therapy (PDT), an emerging anticancer treatment using photoirradiation and photosensitizers. Therefore, the monitoring of ¹O₂ in living cells is highly important for understanding of anti-cancer mechanism of PDT. However, the existing fluorescence probe for the detection of ¹O₂ cannot be used in living cells because of its cell membrane impermeability.
Majima et al. synthesized a new far-red fluorescence probe composed of silicon-containing rhodamine and anthracene moieties, namely Si-DMA, as a fluorophore and a ¹O₂ reactive site, respectively. In the presence of ¹O₂, fluorescence of Si-DMA increases due to endoperoxide formation at the anthracene moiety¹⁾. Among seven different ROS, Si-DMA is able to selectively detect ¹O₂ (Fig. 3). In addition, Si-DMA is able to visualize the generation of ¹O₂ from protoporphyrin IX in mitochondria with 5-aminolevulinic acid (5-ALA), a precursor of heme (Fig. 4).

Fig.1 Cell staining mechanism by Si-DMA

Si-DMA

2 µg x 1

Store at -20 ℃ and protect from light.

• Si-DMA is sensitive to light. Store unused Si-DMA in the bag at -20 ℃.

• Dimethyl sulfoxide (DMSO)
• Hanks’ HEPES buffer or HBSS
• Micropipettes

Preparation of 100 μmol/l Si-DMA DMSO stock solution

Add 36 μl of DMSO to a tube of Si-DMA (2 μg) and dissolve it with pipetting.

• Store the Si-DMA DMSO stock solution at -20 ℃ .
  Si-DMA DMSO stock solution is stable at -20 ℃ for up to a month.

 

Preparation of Si-DMA working solution

Dilute the Si-DMA DMSO stock solution with Hanks’ HEPES buffer to prepare 25-100 nmol/l Si-DMA working solution.

• Protect the solution from light and use it within the same day, since Si-DMA is not stable in Hanks’ HEPES buffer. 

The recommended concentration of Si-DMA working solution for use is 25-100 nmol/l. Once the final concentration of Si-DMA becomes more than 1 μmol/l, Si-DMA may be accumulated to organelle. In addition, the final concentration of Si-DMA becomes more than 5 μmol/l, cytotoxicity may be seen.

Si-DMA staining

1. Prepare cells for the assay.
2. Discard the culture medium and wash the cells with Hanks’ HEPES buffer twice.
3. Add an appropriate volume of Si-DMA working solution.
4. Incubate for 45 minutes at 37 ℃.
5. Discard the supernatant and wash the cells with Hanks’ HEPES buffer twice.
6. Add Hanks’ HEPES buffer and observe the cells under a fluorescence microscope.

Fluorescence microscopic detection of ¹O₂ in HeLa cells treated with 5-aminolevulinic acid (5-ALA)

1. HeLa cells at 2.4×10⁵ cells/ml (200 μl) were seeded on a μ-slide 8 well (Ibidi) in DMEM (10% fetal bovine serum, 1% penicilin-streptmycin) and cultured at in a 5% CO₂ incubator overnight at 37 ℃.
2. The cells were washed with 200 μl of Hanks’ HEPES buffer twice.
3. 5-ALA in Hanks’ HEPES buffer (150 μg/ml, 200 μl) was added to the μ-slide, and the cells were cultured in a 5% CO₂ incubator for 4 hours at 37 ℃.
4. The cells were washed with 200 μl of Hanks’ HEPES buffer twice.
5. Si-DMA working solution (40 nmol/l, 200 μl) was added, and the cells were cultured in a 5% CO₂ incubator for 45 minutes at 37 ℃.
6. The cells were washed with 200 μl of Hanks’ HEPES buffer twice.
7. Hanks’ HEPES buffer (200 μl) were added, and the cells were observed under a fluorescence microscope.

 

Filter (wavelength/band pass)
Fluorescence imaging: 600/50 nm (Ex), 685/50 nm (Em)

Fig.4 Fluorescence imaging of mitochondrial ¹O₂ with Si-DMA in HeLa cells treated with 5-ALA.

Fluorescent of Si-DMA in 5-ALA-treated HeLa cells increased after 2.5 minutes irradiation. It was found that Si-DMA was able to visualize in real time the ¹O₂ generated from protoporphyrin IX in mitochondria.

1. S. Kim, T. Tachikawa, M. Fujitsuka, T. Majima, “Far-Red Fluorescence Probe for Monitoring Singlet Oxygen
   during Photodynamic Therapy”, J. Am. Chem. Soc., 2014, 136 (33), 11707-11715.