Methotrexate (WR19039) is a folate analog, an inhibitor of the dihydrofolate reductase DHFR. Methotrexate has antimetabolic, antitumor, and immunosuppressive activities, and is commonly used in rheumatoid arthritis and various tumors.

DHFR (human):24 nM

方法：6 种儿童白血病和淋巴瘤细胞系 NALM-6、 NALM-16、JURKAT、CEM、RAMOS 和 NAMALWA 用 Methotrexate (0.002-5 μM) 处理 120 h，使用 SRB 方法检测细胞增殖抑制活性。
结果：Methotrexate 对肿瘤细胞的 IC 50 中位数为 78 nM，对六种肿瘤细胞的 IC50 在 33-133 nM 之间。[1]
方法：人卵巢癌细胞 SKOV-3 用 Methotrexate (15-50 μM) 处理 24 h，使用 AO/EtBr probe 检测细胞凋亡情况。
结果：Methotrexate 诱导 SKOV-3 细胞凋亡。[2]

方法：为检测慢性毒性，将 Methotrexate (0.25-6 mg/kg) 腹腔注射给 C57BL/6、DBA/2 和 C3H小鼠，每周五次，持续 12-18 个月。
结果：0.25-2 mg/kg 剂量下的耐受性良好，淋巴组织、睾丸和皮肤中的细胞抑制最小。3-6 mg/kg 剂量下会产生众所周知的急性至亚急性造血和胃肠道损伤，导致早期死亡。[3]
方法：为检测在不同类型风湿性关节炎 (RA) 和多发性硬化症 (MS) 模型中的作用模式，将 Methotrexate (0.1-5 mg/kg) 腹腔注射给不同发病机制的 RA 和 MS 模型，每天一次，持续十四天。
结果：Methotrexate 对 CIA、PIA 等经典 RA 模型及 MS、EAE 模型均有较强的改善作用。Methotrexate 仅在 T 细胞活化之前和依赖于 T 细胞活化的疾病中起作用。[4]

Methotrexate (MTX) is dissolved in DMSO and stored, and then diluted with appropriate media before use[1]. Each cell line is studied in growth inhibition experiments using 96-well microtiter plates. As antifols are schedule dependent, preliminary experiments are aimed at defining the longest duration of exposure that would allow for continuous logarithmic phase growth of cells without changing of the culture media while maintaining a linear relationship between SRB optical density and cell number. Twenty-four hours after cell plating, the cell lines are exposed to the antifol for 120 h (three replicates per experiment). To ensure that a complete sigmoidal survival-concentration curve could be observed, the following drug concentrations are studied: Methotrexate (0.002-5 μM), AMT (0.0001-1 μM), PXD (0.0003-10 μM), TLX (0.0002-0.5 μM). Experiments are repeated at least twice[1].