Methotrexate is a cytotoxic agent used as a chemotherapeutic and immunosuppressant. It is actively taken up by cells and binds to and inhibits dihydrofolate reductase, thereby inhibiting folic acid metabolism and inducing cell cycle arrest. • Green fluorescent CF®488A dye has excitation/emission at 490/515 nm. • Red fluorescent CF®568 dye has excitation/emission at 562/583 nm. • Deep-red fluorescent CF®594 dye has excitation/emission at 593/614 nm. • Far-red fluorescent CF®640R dye has excitation/emission at 642/662 nm.