Fluorescence spectrometer

All procedures were performed according to the Guide for the Care and Use of the Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85-23, revised 1996) and approved by the Institutional Animal Care and Use Committee of Taipei Medical University (Taipei, Taiwan). Male ICR mice were purchased from the Animal Center of the College of Medicine, National Taiwan University (Taipei, Taiwan). Animals were intravenously injected with a single dose of 10 mg/kg CRLX101 following a previous method [29 (link)]. Previous studies have shown the safeties of the empty CD-PEG nanoparticles at a dosage up to 240 mg/kg in mice [43 , 44 (link)]. Mice were sacrificed and blood was collected at 0.5, 1, 3, 12, and 24 hours post-dose and plasma was separated from the blood. Acetonitrile was added to the plasma, and the mixture was centrifuged at 10000 rpm for 10 min to obtain the supernatant. Animals were sacrificed after perfusion. Brain tissues were homogenized and extracted with acetonitrile. To determine the CPT concentration, the fluorescence of CPT in the supernatant from brain tissues and plasma and standard CRLX101 were measured at 370 nm for excitation and 440 nm for emission using a fluorescence spectrometer (PerkinElmer). Concentrations were calculated with a standard curve.

The UV-visible (vis) spectra and fluorescence spectra were obtained using a UV–Vis spectrometer (NanoDrop, Wilmington, DE, USA) and a fluorescence spectrometer (PerkinElmer, Waltham, MA, USA), respectively. Transmission electron microscopy (TEM) observation was performed on a JEM-2100HR transmission electron microscopy (JEOL, Akishima-shi, Japan) operated at 200 kV. Fourier transform infrared (FTIR) spectra were collected using a Tensor 27 FTIR spectrometer (Bruker, Karlsruhe, Germany) in the range 400 to 4,000 cm⁻¹.

An IKA Ultra-Turrax T25 (Staufen im Breisgau, Germany) realized a coarse homogenization of the samples in the extraction solvents. An Hielscher UP200St (Teltow, Germany) was employed for the direct UAE. An UV-Vis Agilent 8453 (Waldbronn, Germany) allowed the determinations of the TPC and RSA, whereas a 55LS Perkin Elmer Fluorescence Spectrometer (Waltham, MA, USA) was employed for the Oxygen Radical Absorbing Capacity (ORAC) determination.

Ethidium bromide uptake was monitored as described earlier (43 (link)). Mab^Wt, Mab^ΔembC, and Mab^ΔCMab were grown to an OD₆₀₀ of 0.6–1.0. Bacteria were collected by centrifugation and resuspended in uptake buffer (PBS, 0.05% Tween 80, pH 7.0) to an OD₆₀₀ of 0.5, followed by the addition of 25 mM glucose for energization. Ethidium bromide (1 μg/mL) was added, followed by real-time measurement in a fluorescence spectrometer (PerkinElmer) with excitation and emission wavelengths set at 520 and 595 nm, respectively. Results were normalized against fluorescence of ethidium bromide and are depicted relative to the highest fluorescence measured. The experiment was performed in triplicate and repeated three times.

Nitric oxide generation was quantitatively measured using DAF-2DA (Calbiochem). Potato leaf samples (0.1 g) were incubated in the dark for 1 h at 25 °C in a mixture containing 10 μM DAF-2DA in 10 mM Tris–HCl buffer (pH 7.2). After incubation, the probes were transferred into 24-well plates (1 mL per well). Fluorescence in the reaction was measured using a spectrofluorometer (Fluorescence Spectrometer Perkin Elmer LS50B, United Kingdom) at 495 nm excitation and 515 nm emission filters. Fluorescence was expressed as arbitrary fluorescence units.