储存
Store at -20℃. This product is stable at -20℃ up to 3 years from the date of receipt .
After reconstitution, the solution is stable at -20℃ for 6 months, at -80℃ for up to 1 year.未开盖的干粉蛋白在 -20°C至-80°C可保存12个月；
复溶之后，蛋白溶液在-20°C及以下可保存3个月，在2-8℃可保存1周。
生物活性
体外活性/In vitro:
1.用Forskolin（0.01-10 µM）处理大鼠肾上腺髓质嗜铬瘤细胞PC12 3-48 h，MTT法检测细胞生长抑制情况。10 μM Forskolin处理后，细胞活力迅速下降，处理6 h后细胞活力下降为88.4%，处理48 h后细胞活力下降为60.5%。[1]
2.用Forskolin（1-100 µM）处理人骨髓瘤细胞U266、H929、INA-6、RPMI 8226和OPM-2 72 h，Flow Cytometry检测细胞死亡情况。Forskolin剂量依赖性诱导人骨髓瘤细胞死亡，其中U266、OPM-2和INA-6比H929和RPMI 8226细胞更敏感。[2]
3.用Forskolin（1-100 µM）处理人IL-2依赖性白血病细胞Kit 225和人白血病细胞MT-2 20 min，ELISA测定cAMP浓度。Forskolin诱导cAMP水平上调，在50-100 μm之间达到最大水平。[3]
体内活性/In vivo:
1.将Forskolin（4-5 mg/kg in PBS/DMSO solution (15:1)）腹腔注射给携带鼠多发性骨髓瘤肿瘤MOPC315的BALB/c nude小鼠，在肿瘤细胞注射后的第2/4/6天给药，检测体内抗肿瘤活性。所有小鼠最终都发生了肿瘤，但Forskolin显著延缓了体内肿瘤的生长。提高cAMP的化合物可能在治疗多发性骨髓瘤方面具有治疗潜力。[4]
2.将Forskolin（50 mg/kg）灌胃给药给STZ诱导糖尿病模型的C57BL/6小鼠，每周一次，持续十二周，研究Forskolin对糖尿病条件下视网膜炎症的影响。与正常对照组相比，糖尿病对照组和Forskolin治疗组的视网膜葡萄糖浓度均增加，但由于葡萄糖转运蛋白1表达下调，Forskolin处理组仅为糖尿病对照组的约68.06%。与正常对照组相比，Forskolin治疗组和糖尿病对照组的ICAM‑1和TNF-α表达上调，但Forskolin处理组的这两种炎症因子表达水平分别为糖尿病对照的68.75%和75.37%。[5]
激酶实验/Kinase Assay:
For Jak3 kinase assays, Fsk-treated MT-2 cells were lysed, clarified, and immunoprecipitated using Jak3 antibody as described above. Kinase reactions were carried out as described previously at 30°C for 20 min. For PKA kinase assays, untreated MT-2 cells were lysed, and Jak3 was immunoprecipitated and bound to PAS beads as described previously. Immunoprecipitated Jak3 was washed with kinase buffer (50 mM Hepes-NaOH (pH 7.4), 10 mM MgCl2, 0.5 mM EGTA, 0.5 mM DTT, 20 μg/ml aprotinin, 10 μg/ml leupeptin, 1 μg/ml pepstatin A) and incubated with 200 μM ATP and purified protein kinase A catalytic subunit (PKAc) as indicated in the figure legends. Kinase reactions were carried out at 32°C for 30 min followed by vigorous washing of the beads with cold kinase wash buffer as described previously. For [γ-32P]ATP radiolabeled kinase assays using recombinant Jak3, Hek293 cells were transfected with wild type (WT) Jak3 or kinase-dead Jak3 K855A using Lipofectamine 2000 according to the manufacturer's instructions. Cells were lysed and immunoprecipitated with Jak3 antibody. Jak3-bound PAS beads were washed three times in cold lysis buffer followed by kinase buffer. Kinase reactions were initiated by adding 10 μCi [γ-32P]ATP, 10 μm unlabeled ATP, and 1 μg of purified PKAc to Jak3-bound PAS bead reaction mixtures. Kinase reactions were performed at 32°C for 30 min. Jak3-bound PAS beads were washed three times in radioimmunoassay buffer (10 mM Tris-HCl, pH 7.4, 75 mM NaCl, 20 mM EDTA, 10 mM EGTA, 20 mM Na4P2O7, 50 mM NaF, 20 mM 2-glycerolphosphate, 1 mM p-nitrophenyl phosphate, 0.1% Triton X-100) and one time in kinase wash buffer. The reactions were stopped by adding 2× SDS-PAGE sample buffer followed by SDS-PAGE. Coomassie stainable Jak3 bands were excised from the PVDF membrane and subjected to phosphoamino acid analysis [2].
