Enspire fluorescence analyzer

Wild-type rat TrxR1 and its variants were dissolved in TE buffer (pH 7.5). The protein concentrations were adjusted to 6.8 μM on the basis of the FAD absorbance at 463 nm. Two hundred microliters (total volume) of these TrxR1 samples were loaded into a flat-bottomed 96-wells black plate (Thermo Fisher Scientific, Waltham, MA, USA) for fluorescence measurements using the EnSpire Fluorescence Analyzer (PerkinElmer, Waltham, MA, USA). The emission spectrum ranging from 250 nm–650 nm was obtained by exiting the sample at a fixed excitation wavelength of 230 nm and subsequently the excitation wavelength was increased by 10-nm intervals up to 510 nm. The data obtained for all excitation wavelengths were used to plot the fluorescence emission spectra in 3D format, with ‘x axis' as the excitation wavelength (Ex), ‘y axis' as the emission wavelength (Em), and the ‘z axis' as fluorescence intensity. After subtracting the TE buffer background, TrxR1 samples have background fluorescence at Em_max=380 nm when excited at Ex_max=280 nm, and three typical flavin fluorescence signaling at Em_max=520 nm when excited at Ex_max=280 nm, Ex_max=370 nm and Ex_max=470 nm.

IAV was diluted to 8 × 10⁵ PFU using 1X Assay buffer (33.3 mM 2-(N-morpholino) ethanesulfonic acid (MES), 4 mM CaCl₂, pH 6.5) containing 0.1% NP-40 and treated with ethyl pheophorbide a (1 µg/mL), ethyl pheophorbide b (1 µg/mL) or zanamivir (1 µM) at room temperature for 45 min. The mixture was subsequently treated with 0.3 mM 2′-(4-Methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUNANA; Sigma-Aldrich), a substrate of NA, and incubated at 37 °C for 1 h. After the reaction was terminated with stop solution (11 mL of absolute ethanol, 2.225 mL of 0.824M NaOH), fluorescence was measured at excitation and emission wavelengths of 355 nm and 460 nm, respectively, using an EnSpire fluorescence analyzer (Perkin Elmer, Waltham, MA, USA).