Varian cary eclipse

As an animal model of lung metastasis, 2 × 10⁵ MC38 cells were inoculated in 100 μl of DMEM via tail vein of the mice. After 22 days, mice were anaesthetized with an i.p injection of Ketamine/xylazine 100 mg/kg and 10 mg/kg body weight, respectively, and transcardially perfused with PBS. A picture of dissected lungs was taken and a GFP fluorescence image was obtained in IVIS Lumina System. Further, lungs were processed for quantification of metastasis by detection of GFP fluorescence. Perfused lungs were homogenized in 2 ml of a hypotonic buffer (20 mM Tris-Cl, pH 7.0) by using a potter. Triton X-100 was added to a final concentration of 0,5 %. After 30 min on ice, the insoluble debris was spun down (10 000 g for 10 min), 10 μl of each supernatant was diluted with Tris-Cl buffer (20 mM Tris-Cl, pH 7.0) to a final volume of 100 μl, and transferred to a quartz cubet. Fluorescence was read on a Varian Cary Eclipse (Varian) at Ex 485 and Em 508. Background levels for subtraction were determined on lungs from mice, which had not been injected with GFP-labeled cells.⁹²

The steady state absorption and photoluminescence spectra of synthesized Au NCs Rh+/Rh− were measured using UV-visible absorption spectrometer Varian Carry 50 (Varian Inc., Belrose, NSW, Australia) and fluorescence spectrometer Varian Cary Eclipse (Varian Inc.). Fluorescence decay measurements were performed using FLS 920 spectrometer (Edinburgh instruments Ltd., Livingston, UK) using the time correlated single photon counting technique. Diode laser (λ = 405 nm, pulse length—<200 ps, pulse repetition rate—100 kHz) was used for excitation of samples. Fluorescence decay was measured at the peak emission wavelength of the Au NCs Rh+/Rh− fluorescence band. All spectral measurements were performed in 1cm pathlength quartz cells (Hellma Optik GmbH, Jena, Germany).
A hydrodynamic diameter of particles was measured using the dynamic light scattering technique. Samples were measured using particle size and zeta potential analyzer Zeta Plus PALS (Brookhaven Inc., Suffolk County, NY, USA).

After aggregation had occurred, each sample was taken out of the 96-well plate and each well was additionally washed with the reaction solution (50 mM sodium phosphate buffer (pH 6.0) with 2 M GuHCl and 100 µM ThT) in order to collect all the fibrils. The samples were then further diluted using the reaction buffer solution to a final volume of 500 µL (5-fold dilution). Samples were then sonicated for 30 s using a Bandelin Sonopuls (Berlin, Germany) Ultrasonic homogenizer, equipped with a MS-72 tip (20% power). Each sample‘s excitation–emission matrix (EEM) was then scanned using a Varian Cary Eclipse (Agilent Technologies, Santa Clara, CA, USA) fluorescence spectrophotometer (excitation wavelength range was from 435 to 465 nm, emission wavelength range—from 460 to 500 nm; both excitation and emission slit widths were 5 nm) at 25 °C. Absorbance spectra were acquired from 300 to 600 nm using a Shimadzu (Kyoto, Japan) UV-1800 spectrophotometer. EEMs were then corrected for the inner filter effect and their signal intensity centre of mass was calculated as described previously [48 (link)]. All EEM data is available as Supplementary Material. Sample maximum fluorescence intensity distributions were compared using a one-way ANOVA Bonferroni means comparison with a significance level of 0.01 (Origin 2018 software, OriginLab Corporation, Northampton, MA, USA).

The method for the PS80 quantification was adapted from Zheng et al. [27 (link)]. Samples were diluted 1:4 or 1:10 with DPBS and subsequently heated for 5 min at 99 °C. Afterwards, samples were centrifuged for 5 min at 25,700×g. 190 µL supernatant were mixed with 10 µL of 1 mM bis-ANS and vortexed for 5 s. 60 µL of each sample were analysed in a Varian Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) using a quartz cuvette at 380 nm excitation and 500 nm emission with both slits set to 5 nm. A calibration curve of PS80 in DPBS allowed the quantification of PS80 between 0.005 and 0.15 mg/mL (R² = 0.9988).

The fluorescence quenching assay was performed according to the protocol described earlier [37 (link)]. Fluorescence spectra were recorded with a Varian Cary Eclipse fluorescence spectrofluorometer (Agilent Technologies, Singapore) equipped with an electro-thermal temperature controller. The intrinsic fluorescence emission spectra of native and recombinant OVGP1 were recorded from 290 to 500 nm upon excitation at 280 nm wavelength using a quartz cuvette of 1.0-cm path length. Excitation and emission slits were maintained at 5 nm and the scan speed was set to 100 nm/min. All spectra were recorded at a constant temperature of 298 K. Standard reaction mixtures were prepared using 10 μM solution of protein in 25 mM phosphate buffer saline, pH 7.2 to a final volume of 1 ml. Different sugar ligands i.e. GlcNAc (N-acetyl glucosamine), GalNAc (N-acetyl galactosamine), Man (mannose), (GlcNAc)₂, (GlcNAc)₄ and (GlcNAc)₄ were prepared at different concentrations (5, 10 and 15 mM), pre-incubated with a fixed concentration (10 μM) of native and recombinant OVGP1 for about 1 h and spectra were obtained. Appropriate blanks corresponding to the buffer were subtracted to correct the background emission of fluorescence.

Fluorescence emission spectra were recorded on a Varian Cary Eclipse fluorescence spectrometer (Agilent Technologies, Santa Clara, CA, USA). λ_exci(pyrene) = 330 nm.