Tetrahydrobiopterin  (Synonyms: (Rac)-Sapropterin)

Microglia cell line (SIM-A9) is used and cultured. Briefly, microglial cells (800, 000 cells per well) are cultured in 6-well plates with DMEM/F12 (1:1) supplementing with 10% fetal bovine serum (FBS), 5% of horse serum (HS), and 1% penicillin/streptomycin. After 24 h, the cells are starved with DMEM/F12 (1:1) free of FBS and HS for 6 h. Then, microglial cells cultures in presence or absence of 100 μM of Tetrahydrobiopterin are exposed to hyperoxia (75% oxygen and 25% nitrogen) in a modular incubator chamber and maintained in a humidified CO₂ incubator at 37 °C for 24 h. Microglial cells in matching controls are incubated at 37 °C in an incubator with 95% air and 5% CO₂ and collected at the same time point. Cell lysates are quickly processed for RNA. The conditioning media is stored at -80 and later used in choroidal explant assay^[1].

MCE はこれらの方法の精度を確認していません。 こちらは参照専用です。

Mice pups are exposed with their mothers in a 75% oxygen environment from postnatal day 7 to P9 using oxycycler to induce retinal vaso-obliteration (VO). Animals are anesthetized and injected intravitreally at P7 with 100 μM of Tetrahydrobiopterin or vehicle (sterile PBS 1×) using a syringe equipped with 50-gauge glass capillary. At P9, mice pups are sacrificed and retinas are dissected and stained overnight at 4 °C with fluorescein-labeled Griffonia Simplicifolia Lectin 1 (GSL 1), isolectin B4 (1:100) with 1 mM CaCl₂ in PBS. Quantification of VO is assessed using the computer software^[1].

MCE はこれらの方法の精度を確認していません。 こちらは参照専用です。

• [1]. Rivera JC, et al. Tetrahydrobiopterin (BH4) deficiency is associated with augmented inflammation and microvascular degeneration in the retina. J Neuroinflammation. 2017 Sep 6;14(1):181.  [Content Brief]