Hplc grade chloroform

Lipid and polar metabolites were extracted with a 1:2:0.8 mixture of chloroform: MeOH: H₂O, following a modified version of a published protocol [35 (link)]. HPLC grade chloroform and methanol were from Sigma-Aldrich. All the steps were done using glassware, to avoid polymers contamination. The extractions were performed in 15 mL Kimble^™ Kontes^™ KIMAX^™ Reusable High Strength Centrifuge Tubes from Fisher Scientific. Half of the original protocol volume values were used. For extractions headed to GC-MS analysis, 50 μL of ribitol internal standard (10 mg/mL) were added. 3 mL of the polar and 3 mL of the lipid phase were collected per sample. The polar phase was dried in SpeedVac (ThermoFisher Scientific). The lipid phase was dried overnight in a fume hood. For ESI-MS experiments, but not for GC-MS, the dried polar phases were re-suspended in 500 μL of MeOH and the lipid phases were re-suspended in 500 μL 1:1 chloroform: MeOH. All extracts were stored at -20 ^oC for analysis within 48 hours.

To extract the PHA, the dried biomass obtained from each flask was placed in an extraction thimble, covered with cotton wool, transferred to a Soxhlet extraction set up with HPLC-grade chloroform (Sigma-Aldrich, St. Louis, MO, USA), and left to run for 48 h. As part of a chloroform–biopolymer mixture, the PHA extracted was collected. Using a rotary evaporator set to 50 °C, leftover chloroform present in the mixture was evaporated, leaving behind the extracted PHA gathered in a 250 mL round-bottomed flask. In order to further purify the obtained biopolymer, PHA was precipitated in n-hexane. The precipitation was conducted in the round-bottomed flask. The flask was swirled gently to dislodge any PHA stuck to the flask, and the dispersion formed afterwards was filtered with a filter paper (Whatman No. 1 paper, Whatman Laboratory, Cardiff UK) to separate the polymer from the hexane solution. The purified polymer was left in a fume cupboard to dry, and later formed a thin PHA film. The PHA yield was determined afterwards using Equation (1): $$\mathrm{PHA} \mathrm{yield} \left(\%\right)=\left(\frac{\mathrm{Weight} \mathrm{of} \mathrm{extracted} \mathrm{polymer} \left(\mathrm{WPHA}\right)}{\mathrm{Cell} \mathrm{dry} \mathrm{weight} \left(\mathrm{CDW}\right)}\right)\times 100$$

LC-MS grade water, methanol, propan-2-ol and acetonitrile (ACN), HPLC grade chloroform, reagent grade formic acid, ammonium formate, and sodium acetate were purchased from Sigma Aldrich (Milan, Italy). Various wakame seaweed samples were bought, as dried chunks, from local grocery stores.

2,5-Dihydroxybenzoic acid (DHB), norharmane, Fluoroshield with DAPI, Monoclonal Anti-Actin α-Smooth Muscle antibody, LC-MS grade methanol, HPLC grade chloroform, Bouin’s solution, Triton X-100, Tween 20, poly-L-lysine solution 0.1% and Peel-a-Way embedding molds (truncated, 12 × 12 × 20 mm) were purchased from Sigma-Aldrich, Saint Louis, MO, USA. Histology-grade solvents, Eosin G or Y (alcoholic solution 0.5%) and Masson’s Trichrome staining kit were purchased from DiaPath S.p.A., Martinengo, Italy, while QA-Agarose low melting point was obtained from MP Biomedical, Santa Ana, CA, USA. Harris hematoxylin, phosphate buffer saline (PBS) tablets, bovine serum albumin (BSA), SuperFrost Plus glass slides and cover slides were purchased from VWR International, LLC, Radnor, PA, USA. Trifluoroacetic acid (TFA), Antihuman CD68 antibody and AlexaFluor 568 goat anti mouse secondary antibody were purchased from Life Technologies-Invitrogen, Carlsbad, CA, USA. LC-MS grade water was purchased from Thermo Fisher Scientific, Rockford, IL, USA, while ITO coated glass slides for MALDI were obtained from Bruker Daltonics, Billerica, MA, USA. Paraformaldehyde 4% solution was purchased from Alfa Aesar, Haverhill, MA, USA.

PHA extraction was carried out as described previously [31 (link)]. Shake flask experiments were left to run for 48 h after which microbial experiments were stopped and cultures were centrifuged in a Hermle Labortechnik (Wehingen, Germany) Z300K centrifuge for 15 min at 6000 rpm. Supernatants were discarded and the biomass was obtained and frozen overnight at −20 °C. This was followed by lyophilization using an Edwards Modulyo freeze-drier (Crawley, UK) for 72 h at a temperature of −40 °C and at a pressure of 5 mBar. The weights of the dry biomass were obtained and recorded as cell dry weight (CDW). Lyophilized biomass was then transferred into extraction thimbles and PHA was extracted by Soxhlet extraction with HPLC grade chloroform (Sigma Aldrich) running for 5 h. The hot solution of polymer/chloroform was concentrated by evaporation, followed by PHA precipitation in n-hexane (Sigma Aldrich). Precipitated polymer was separated from solution by filtration (Watman No. 1 paper, Sigma Aldrich) and rinsed further in chloroform to remove the excess wax materials before air-drying. The weight of the polymer (W_PHA) was recorded and the percentage of PHA (wt/wt) synthesized by R. eutropha H16 was calculated using the equation:
$PHA\%=\frac{W_{PHA}}{CDW}\times 100$

Indole-3-acetic acid and serotonin HCl were purchased from Alta Aesar (Ward Hill, MA, USA). All other tryptophan metabolites, HPLC grade chloroform and indole-d6 were purchased from Sigma–Aldrich (St. Louis, MO, USA) or Toronto Research Chemicals (North York, ON, Canada). All tryptophan metabolites were of the highest grade available (>96%). Indole was re-purified and verified by NMR as previously described.^{32 (link)} Analytical grade sodium hydroxide and sodium bicarbonate, as well as LC/MS grade solvents including methanol and acetonitrile, were purchased from Fisher Scientific (Hampton, NH, USA). The stock solutions of all reference standards for liquid chromatography were prepared in 10% acetonitrile (v/v) containing 1 µM chloropropamide (internal standard). The mixed standard solutions were gradually diluted with 10% acetonitrile (v/v) containing 1 µM chloropropamide for generating the calibration curves. The stock solutions of reference standards for GC were prepared in chloroform containing 30 µM indole-d6 (internal standard).