TMPyP4 tosylate

Cell lines

Preparation Method

Telomerase Assay
Telomerase activity was assayed using the TRAPeze XL Telomerase Detection Kit. To determine whether TMPyP4 tosylate directly inhibits telomerase activity in vitro assay, the TRAPeze XL reaction mixture (50µl) containing the lysates of 10³-10⁴ osteosarcoma cells were incubated for 30min at 30°C. Then, perform a four-step PCR at 94°C/30s, 59°C/30s, 72°C/1min for 36 cycles followed by a 72°C/3min extension step and then at 55°C/25min, concluding with a 4°C incubation. The PCR products were analyzed with fluorescence plate readers.
Measurement of Apoptosis by Fluorescence Microscopy
Apoptotic and dead cells after treatment with TMPyP4 tosylate were determined using the Annexin V-FITC Apoptosis Detection kit. The cells were incubated in the presence of 50 or 100µM TMPyP4 tosylate for 96h. Then, 1 × 10⁵ cells were collected by centrifugation, resuspended cells in 500µl of 1× binding buffer, and added 5µl of Annexin V-FITC and 5 µl of propidium iodide. The cells were incubated 5min in the dark at room temperature and stained cells were analyzed by fluorescence microscope. The experiments were performed twice. Two independent observers evaluated stained sections and the rates of stained cells were determined based on average values. For the evaluation of stained cells, we examined at least 700 osteosarcoma cells to determine whether their cell membranes were positive for Annexin V staining.

Reaction Conditions

Applications

Animal models

female BALB/c mice

Preparation Method

Establishment of Infection Model
To establish the infection, 3×10⁷ yeast forms of C. albicans in a volume of 30µL of phosphate-buffered saline (PBS) were injected intradermally into the ears of anesthetized female BALB/c mice.
In Vivo Photodynamic Therapy (PDT) Conditions
Two days post-infection, a topical application of 0.3mg/mL TMPyP4 tosylate (50µL) in either a glycerol/water (2:3) or ethanol/glycerol/water (2:2:1) vehicle was applied to the dorsal surface of the ear. Control ears were infected but left untreated. 30min after the application of the photosensitizer, the ear was gently cleaned with a cotton swab soaked in phosphate-buffered saline. Subsequently, the dorsal surface of the ear was irradiated with 514nm light from an argon-ion laser delivered through a GRIN-lens-terminated multimode fiber. The treatment fluence was 90J/cm², and the irradiance was 50mW/cm².
Assay of In Vivo Phototoxicity

Dosage form

Applications

References:
[1] Fujimori J, Matsuo T, Shimose S, et al. Antitumor effects of telomerase inhibitor TMPyP4 in osteosarcoma cell lines. J Orthop Res. 2011 Nov;29(11):1707-11.
[2] Mitra S, Haidaris CG, Snell SB, et al. Effective photosensitization and selectivity in vivo of Candida Albicans by meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate. Lasers Surg Med. 2011 Apr;43(4):324-32.