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, BGC-823 cells:0.11 μM, CEM cells:33-133 nM, COLO 205 cells:3.27 μM, A549 cells:0.013 μM, 5637 cells:0.016 μM, HEK293 cells:1.27 μM, JURKAT cells:33-133 nM, A427 cells:5.52 μM, DAN-G cells:0.077 μM, NALM-6 cells:33-133 nM, NALM-16 cells:33-133 nM, HBL-100 cells:0.04 μM, NAMALWA cells:33-133 nM, Ramos cells:33-133 nM

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].