TRFS-green is a highly selective off−on fluorescent probe for imaging selenoprotein thioredoxin reductase (TrxR) in living cells. TRFS-green has the maximum absorbance at around 373 nm. After it is activated by TrxR , the maximum absorbance shifts to around 440 nm. In Vitro TRFS-green displays a green fluorescence off−on change induced by TrxR-mediated disulfide cleavage and subsequent intramolecular cyclization to liberate the masked naphthalimide fluorophore. TRFS-green gives a signal at ~480 nm when excited at 377 nm. Pretreatment of living cells with TrxR inhibitors in a dose-dependent manner can inhibit the fluorescence signal of TRFS-green in living cells, thereby facilitating the development of living cell-based screening assays for TrxR inhibitors. TRFS-green shows no response to GSH or Cys, the predominant thiols in cells, or the endogenous reducing agents, such as vitamin C and NADPH. Measure TrxR activity in crude cell extracts: Incubate cell lysates with TRFS-green for an appropriate time (usually 1-2 hours), and measure fluorescence intensity by fluorimeter (Em= 540 nm, Ex=440 nm). It is also possible to monitor the time-dependent increase in fluorescence (Em=540 nm, Ex=440 nm) over a given period of time. Cells treated with TRFS-green (10 μM; 2-4 h) can be observed through the green fluorescent channel of the microscope. Note: In order to obtain a clear fluorescence image, it is recommended to wash the cells with PBS or fresh medium immediately before taking pictures to remove residual TRFS-green. TRFS-green is the first fluorescent probe of TrxR with a green emission. However, the response rate of TRFS-green to the enzyme is slow, and it takes more than 2 h to reach the maximal fluorescence signal even using tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent. MCE has not independently confirmed the accuracy of these methods. They are for reference only. Form:Solid