Gf c glass microfibre filters

Prior to carrying out the microplastics extraction and characterization, the digestive system of each specimen was rinsed with pre-filtered (0.22 μm) deionized water and centrifuged to eliminate alcohol used to store them. Subsequently each pellet was incubated with 10% of KOH (w/v) solution prepared using KOH pellets (Sigma-Aldrich, Saint-Quentin-Fallavier, France) and double-distilled water. Then they were placed on an agitation plate (IKA RT15, Staufen, Germany) set at 300 rpm and 60 ± 1 °C for 24 h. After digestion, all samples were filtered on 90 mm diameter GF/C glass microfibre filters (Whatman, Velizy-Villacoublay, France) using a vacuum system. Filters were then placed in closed Petri dishes until subsequent analysis. For the first characterisation, filters were observed under a stereomicroscope (Nikon SMZ25, Tokyo, Japan), allowing the identification of plastic particles. Items with characteristics similar to plastic polymers were characterized by size (< 100 µm; 100–500 µm; ˃500 µm) and colour. Filtered fragments and fibres were then rinsed in distilled water and mounted on double-sided adhesive carbon tabs on aluminium SEM stubs for successive SEM/EDX analyses.

Binding experiments were conducted as in [13 (link)]. Briefly FPR1-human embryonic kidney (HEK)–or ALX-HEK—transfected cells (1x10⁶ cells/ml) were incubated with increasing concentrations of cold CR-AnxA1_2-50, CR-AnxA1_2-48 (0.1nM-10μM) or PBS and a fixed concentration (50nM) of [¹²⁵I-Tyr]-Ac2-26 for (1h, 4°C). Bound and unbound tracer were separated by filtration through Whatman GF/C glass microfibre filters (Kent, United Kingdom) using a vacuum manifold, and the amount of tracer bound to the cells was quantified by counting filters in a gamma-counter.
Receptor activation was monitored using the β-Arrestin PathHunter system (Discoverx) with experiments conducted as in [17 ] using HEK cells stably overexpressing recombinant human ALX receptors tagged with a prolink label of β-galactosidase (β-gal) and β-arrestin linked to the enzyme acceptor fragment of β-gal. In brief, cells were plated in 96-well plates (20,000 cells/well) 24 h before initiating experiments. CR-ANXA1_2-50 and CR-AnxA1_2-48 were incubated with cells (60 min, 37°C), and receptor activation was determined by measuring chemiluminescence using the PathHunter detection kit (Discoverx).