Infinite 200 pro nanoquant

DOX was encapsulated into the iEDNs using the phosphate-gradient method [20 (link)]. Briefly, iEDNs (1 mg) were prepared in 300 mM ammonium phosphate buffer (pH 7.4) and then passed through a PD-10 column equilibrated with the HEPES buffer (25 mM HEPES, pH 7.4). An aliquot of DOX solution (5 mg/mL; Sigma Aldrich, St. Louis, MI, USA) was added to the prepared iEDNs (1:8 weight ratio of DOX and EDN) and incubated for 48 h at 37 °C. Free DOX was removed by gel filtration using a PD-10 column, equilibrated with HEPES buffer, and the fractions of iEDNs-DOX were concentrated by centrifugation in a 30 K Amicon tube (Millipore) for 10 min at 3500× g. The efficiency of DOX encapsulation was analyzed by measuring the absorbance at 480 nm using an Infinite 200 Pro NanoQuant (TECAN Group Ltd., Mannedorf, Switzerland).
The cumulative release of DOX from EDNs-DOX and iEDNs-DOX was evaluated by the dialysis method using a 3.5 kDa DiaEasy Dialyzer (Biovision Inc., Mipitas, CA, USA). Free DOX, EDNs-DOX, and iEDNs-DOX (100 μg of DOX or equivalent) were added to the dialysis tubes and dialyzed in 1 L of HEPES buffer (25 mM, pH 7.4) with constant stirring for 48 h. The amount of remaining DOX was measured using an Infinite 200 Pro NanoQuant (TECAN) at a wavelength of 480 nm.

According to the manufacturer's instructions, the concentration of peptides yielded after digestion was quantified using the MicroBCA assay (ThermoFisher, USA). Absorbance was quantified on a colorimetric plate reader at 540 nm (Infinite 200 Pro NanoQuant—Tecan, Switzerland), converted to protein concentration using standards provided by the kit.
Glycosaminoglycans (GAGs) in solution were quantified using dimethyldimethylene blue staining (DMMB), as previously described [31 (link)]. A digested ECM (20 μL) sample was added to the DMMB solution (200 μL). Absorbance was quantified on a colorimetric plate reader at 535 nm (Infinite 200 Pro NanoQuant—Tecan, Switzerland).
In order to evaluate the presence of protein secondary structures, samples of digested ECM were analyzed using circular dichroism (JW720, Jasco, Japan) between 190 nm ≤ λ ≤ 300 nm, 20 nm/min, and accumulation was set in 3.

Mussels collected from sites 3 and 4 were chosen for 18S rDNA metabarcoding analysis due to the opposite characteristics of their habitats of origin. Individuals analyzed from site 3 were designated as MF (Mussel Farm) and from site 4 as WT. Nucleic acids were extracted from gut samples using PureLink microbiome DNA Purification kit (Invitrogen, Waltham, MA, USA) according to the manufacturer´s instructions. DNA concentration and purity were analyzed with an Infinite^® 200 PRO Nanoquant (Tecan Group Ltd., Männedorf, Switzerland), Qubit 3.0 (Thermo Scientific, Waltham, MA, USA), and a Bioanalyzer Instrument (Agilent Technologies, Santa Clara, CA, USA) with GQN ≥7.
Sequencing was performed on Illumina Miseq (Illumina, San Diego, CA, USA) using Nextera XT v3 600 cycle kit at Fraunhofer Foundation (Santiago, Chile). Samples were amplified by dual-indexing Illumina fusion primers that targeted the 18S rDNA V4 region for eukaryotes [58 (link)]. The manufacturer’s recommended protocol was used to perform the sequencing reaction on Illumina Miseq platform. Sequence reads data were archived at NCBI Sequence Read Archive (SRA) with the BioProject number: PRJNA762938.