Fl winlab software

The efficiency of fluorescence labeling with Cy5-dUTP was analysed with the PerkinElmer LS55 using FL Winlab software (PerkinElmer) and FLUOstar Omega (BMG Labtech). Using LS55 fluorescence spectrometer, purified LAMP products were diluted in a ratio of 1:16 (v/v) with a final volume of 96 µl and analysed using the following parameters: (i) 630 nm excitation and emission spectrum ranging from 600 to 800 nm; (ii) Emslit/Exslit = 10 nm; (iii) scan speed = 100 nm/min. All measurements were conducted as triplicates.
Using FLUOstar Omega, 30 µl purified samples were analysed without dilution using 620 nm excitation and 680 nm emission filter.
For the standard calibration line, free Cy5-dUTP was taken at a dilution range of 7.8E-03 mM to 2.5E-01 mM.

Plasma levels of cytokines were assessed by enzyme-linked immunosorbent assays (ELISA). Assays were carried out according to the manufacturer’s instructions (all kits from eBioscience). Also, the level of cytokines was determined in protein extracts from hippocampi of FMT-treated mice using Procarta Plex Analyst 1.0 (eBioscience) following instructions provided by the manufacturer. Intestinal permeability was assessed using a method described previously [73 (link)]. Animals were orally delivered with 0.5 mL of PBS containing 25 mg of FITC-labelled dextran (FD4; Sigma-Aldrich), and blood samples were collected after 45 min. Plasma concentration of FD4 was assessed using fluorescence spectrometer (LS 55 conducted with FL WinLab software, PerkinElmer, USA) at an excitation wavelength of 490 nm and emission wavelength of 520 nm.

IR spectroscopy analysis was performed by using Nicolet Omnic 8.0 software (Thermo Nicolet Co., USA). Nuclear magnetic resonance spectroscopy (CPMAS¹³C-NMR) was determined by using MestReNova professional analysis software (Mestrelab Research SL, Santiago de Compostela, Spain). Fluorescence spectra were collected using FL WinLab software (Perkin-Elmer). After analyzing and extracting the source data, Microsoft Office Excel 2010 and Origin 8.0 software were used for data processing and preparing figures. The Origin plot was used to draw the data in the “Available Data” as a fit curve fit, and SPSS 19.0 statistical analysis software was used for the significant difference tests (Duncan method).

The fluorescence measurements were carried out on an LS 55 fluorescence spectrometer from Perkin Elmer with a FL Winlab software. Polymersomes loaded with TPyCP were measured in a 1 cm path length quartz cuvette. A wavelength of 424 nm was used to excite in the Soret band and the emission was monitored at 580 nm. Excitation and emission slits were set at 7.5 nm.

Nitric oxide release was determined using the fluorescent probe 4,5-diaminofluorescein (DAF-2), as previously described [19] (link). Briefly, the second, third and fourth branches of mesenteric artery were divided in two experimental groups: control and tranilast-incubated segments (100 µmol/L, 1 hour). After an equilibration period of 30 min in HEPES (in mmol/L: 119 NaCl, 20 HEPES, 46 KCl, 1 MgSO₄.7H₂O, 0.15 Na₂HPO₄.12H₂O, 0.4 KH₂PO₄, 5 NaHCO₃, 1.2 CaCl₂.2H₂O, 5.2 glucose) at 37°C, arteries were incubated with 2 µmol/L DAF-2 for 45 min and medium was collected to measure basal NO release. Once the organ bath was refilled, ACh-induced NO release was measured after an ACh concentration-curve (0.1 nmol/L - 3 µmol/L) was applied at 2-min intervals each dose. The fluorescence of the medium was measured at room temperature using a spectrofluorimeter (LS50 Perkin Elmer Instruments, FL WINLAB Software) with excitation wavelength set at 492 nm and emission wavelength at 515 nm. The stimulated NO release was calculated by subtracting the basal NO release from that evoked by ACh. Also, blank measurement samples were collected from medium without mesenteric segments in order to subtract background emission. Some assays were performed in the presence of L-NAME in order to assure assay specificity. The amount of NO released was expressed as arbitrary units/mg tissue.