Phosphate buffered saline (pbs)

To test the pH stability, phage lysates were diluted hundredfold in LB medium adjusted to pH values ranging from 1 to 12. After incubation for the indicated time at specified temperatures, phages were serially diluted in SM buffer. Viable phages were determined by soft-agar overlay assays, using E28 as host strain. Data are presented as mean of duplicate determinations.
Stability during storage of bacteriophages was examined in PBS (2.7 mM KCl, 8 mM Na₂HPO₄, 1.7 mM NaH₂PO₄, pH 7.0), PBS supplemented with 0.1 or 5 g/L NaCl (all components from Carl Roth GmbH & Co KG, Karlsruhe, Germany), or LB medium as described above.

Prior to dissection, the animals were anaesthetised with CO₂. Dissection was performed in phosphate buffer solution (PBS) (Carl Roth GmbH & Co KG, Karlsruhe, Germany). The specimens were briefly subjected to small amounts of Triton X-100 (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), to remove air bubbles trapped on the surface by decreasing water surface tension. Triton X-100 then was washed repeatedly with the PBS to fully remove traces of Triton X-100. Microscopical observations were made after transfer of antennae or antennal fragments to glycerine (Carl Roth GmbH & Co. KG, Karlsruhe, Germany).

After incubation, endothelial cells attached to the filter membranes were washed with PBS (Gibco, Germany) and fixed with a mixture of methanol/ethanol (2∶1) at room temperature for 20 min. Cells were washed and stained with antibodies recognizing different tight junction proteins (zonula occludens protein-1, occludin (both Zymed Laboratories, CA, USA), claudin-5 (Abcam, UK)) and the corresponding secondary antibodies (Alexa fluor 546; Molecular Probes, CA, USA). All antibodies were diluted in 1% bovine serum albumin (Roth, Germany) in PBS. Nuclei were stained with Hoechst 33342 dye (Sigma-Aldrich, USA). The filter membranes were embedded with GelMount (Biomeda, Natutec, Germany) and analyzed via fluorescence microscopy (Olympus IX71 with Delta Vision system, Applied Precision, USA).

After the indicated treatments, SkMs were seeded on chamber slides (3500 cells/cm²). After 24 h, the supernatant was removed, and the cells were washed with PBS (PAN Biotech, Aidenbach, Germany), covered with 4% formaldehyde solution (27248, Otto Fischar GmbH, Saarbrücken, Germany) for 10 min and rinsed with 0.1% Tween 20 (9127.1, Carl Roth GmbH, Karlsruhe, Germany) in PBS 3 times. Fixed SkMs were permeabilized for 10 min using 0.1% Triton X-100 (T8787, Merck KGaA, Darmstadt, Deutschland) in PBS and incubated with 1:200 diluted skeletal muscle actin antibody (MA5-12542, Thermo Fisher Scientific, Waltham, MA, USA) and the VectaFluor Amplified Kit (DK2488, Vector Laboratories, Burlingame, CA, USA) or with 1:200 diluted desmin antibody (ab227651, Abcam, Cambridge, UK) and the VectaFluor Amplified Kit (DK1594, Vector Laboratories) for staining, according to the manufacturer’s protocol. After staining, cells were covered with a coverslip in mounting medium with DAPI (DK2488, Vector Laboratories).

Eosinophils were purified from EDTA blood by immunomagnetic negative selection (EasySep™ Direct Human Eosinophil Isolation Kit, Stem Cell Technologies, Grenoble, France) using half the amounts of RapidSpheres and Isolation Cocktail recommended in the manufacturer’s protocol. The viability was determined by flow cytometry analysis using 7-AAD (Miltenyi Biotec, Bergisch Gladbach, Germany) and was found to be ≥99%. Human eosinophils were identified using CD15-PB, CD16-APC-A750 (Beckman Coulter, Brea, CA, USA), and CD193-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany) antibodies. Isolated eosinophils had a median purity of 95.8%. For cytospins, cells were washed with PBS (Carl Roth, Karlsruhe, Germany) and centrifuged onto object slides using the Cytospin 4 Centrifuge (Thermo Scientific, Darmstadt, Germany).

Most of PETases can break the ester bond of BHET to produce TPA and ethylene glycol (EG) with MHET as intermediate product^{17 (link),18 ,38 (link)}. Therefore, BHET is often selected as the representative substance to study the activity of PETase^{17 (link),39 (link)}. In this study, BHET and in vitro expressed PETase candidates were incubated in phosphate-buffered saline (PBS, Carl Roth, Karlsruhe, Germany) at indicated temperatures for 4 days. The yields of TPA and MHET were quantified with UltiMate 3000 UHPLC system from Thermo Scientific with a Triart C18 column (YMC Europe GmbH, Dinslaken, Germany) and a VWD-3400 detector (Thermo Scientific)¹⁹ . The data were analyzed with Compass HyStar software package from Bruker (Billerica, MA, USA).