Fluorescence spectrophotometer

A Perkin Elmer fluorescence spectrophotometer (Perkin–Elmer Corporation, Connecticut, USA) instrument and 0.5 cm × 0.5 cm quartz cuvettes were used to obtain fluorescence spectra of the recombinant G4LZI3 protein based on the method described earlier, with modifications.^{19 (link)}
The spectra were documented at a protein concentration of 0.10 mg/mL in 50 mM Tris-HCl buffer (pH 7.0) at room temperature. Tris-HCl buffer was used as blank, while the excitation wavelength was 270 nm. The intrinsic fluorescence emission scanning was documented within the wavelength of 200-450 nm.

A Zetasizer Nano-ZS90 (Malvern, UK) was used to measure the size and zeta potential of samples. Scanning electron microscopy (SEM, Quanta 200 FEG, Portland, OR, USA), transmission electron microscopy (TEM, Tecnai-F30, Portland, OR, USA), and atomic force microscope (AFM) were used to study the morphology of samples. The drug release rate and loading efficiency were obtained using fluorescence spectrophotometer (PerkinElmer, Waltham, MA, USA). X-ray powder diffraction (XRD) measurements were recorded by D2 PHASER Bruker AXS GmbH (Karlsruhe, Baden-Württemberg, Germany).

Lipofectamine 2000 transfection reagent (Thermo, Waltham, MA, USA) and DNA were respectively diluted into equal amount of Opti-MEM medium (Thermo, Waltham, MA, USA). Diluted Lipofectamine 2000 transfection reagent was mixed with DNA, and then incubated for 20 min at room temperature. After that, the complex was added to cells, and incubated for 6 h at 37 °C. Thus, BV2 microglial cells were co-transfected with 12 μg Renilla plasmids and 12 μg NF-κB plasmids transiently with Lipofectamine 2000 transfection reagent (60 μL) for 48 h. BV2 microglial cells were subsequently treated with gradient concentration of CTL (0, 2.5, 5, and 10 μM) and 1 μg/mL of LPS or vehicle for overnight. Soon thereafter, cells were lysed, and dual luciferase reporter gene assay kit (Bioassaysys, Hayward, CA, USA) was used for detecting NF-κB reporter gene luciferase activity, and analyzed on a fluorescence spectrophotometer (PerkinElmer, Waltham, MA, USA).

Generation of liposomes mimicking the membrane composition of the endoplasmic reticulum (ER) and plasma membrane, and liposome disruption assays, was carried out as described [31 (link), 32 (link)]. All lipids were procured from Avanti Polar Lipids (Alabaster, AL, USA). For the assay, GST-6K, at a concentration of 0.1 μM to 10 μM, was incubated in the presence of dye-encapsulated liposomes for 30 minutes at 25°C, and end-point fluorescence was monitored at 585 nm. Purified GST was used as a control in all experiments. Experiments were carried out in triplicates on a Perkin Elmer fluorescence spectrophotometer using a quartz cuvette.

Mice were fasted overnight before gavage with FITC-dextran (600 mg/kg; ~4000 MW, Sigma-Aldrich), with food supply restored 2-hrs post-gavage. 2-hrs later, serum was separated from blood samples by centrifugation (2300 xg, 5mins, room temperature (RT)), and diluted 1:1 in PBS in a 96-well plate. Serum FITC-dextran concentrations were determined using a fluorescence spectrophotometer (Perkin Elmer), with serum samples from non-FITC-dextran-gavaged mice used as baseline. Standard curves were generated using known concentrations of FITC-dextran diluted in control serum. Concentrations were determined using linear regression of a standard curve.

Fluorescence measurements were recorded at room temperature with a Perkin Elmer fluorescence spectrophotometer. The excitation wavelength was 285 nm, and the emission and excitation slit widths were set at 6 and 3 nm, respectively. The fluorescence spectra were obtained in the range of 290–550 nm at an integration time of 1.0 s/nm. The measurements were performed by adding small aliquots of amino acid derivatives with a 0.0025% concentration.