Standard reagent

UTP, ionophores, and standard reagents were from Sigma-Aldrich (St Louis, MO, USA). DFU was from Merck (Rahway, NJ, USA). Prostaglandin E₂ was from Cayman Chemical (Ann Arbor, MI, USA). Gö6976, Gö6983, Gö6850, and inhibitors of standard signaling pathways were from Calbiochem (San Diego, CA, USA). Fura-2/AM was from Invitrogen (Carlsbad, CA, USA). Cytokines were from PeproTech (London, UK). Antibodies against P2Y2, P2Y4, and P2Y6 receptors were from Alomone Labs (Jerusalem, Israel) and other antibodies were from Santa Cruz Biotech (Santa Cruz, CA, USA), from Cell Signaling (Danvers, MA, USA), or from the sources previously described [22 ]. Reagents for electrophoresis were from Bio-Rad (Hercules, CA, USA) and Sigma-Aldrich. Tissue culture dishes were from Falcon (Lincoln Park, NJ, USA) and culture media were from Invitrogen.

The cyclotron parameters used in the production of carrier-added [¹⁸F]F₂ are summarized in Supplemental Table 1. The target gas ([¹⁸O]O₂ > 98%) used for the first bombardment on the cyclotron is obtained from Campro Scientific (Berlin, Germany). The synthesis is carried out using a custom-build synthesis platform (see Supplemental Figs. 1 and 2). An overall synthesis flowchart can be found in Supplemental Fig. 3. The precursor 2 for radiolabeling is obtained from ABX advanced biochemical compounds (Radeberg, Germany), standard reagents are obtained from Sigma-Aldrich, sterile solutions from the Aarhus University Hospital Pharmacy (Aarhus, Denmark), SPE cartridges and sterile filters from Waters, and the high-performance liquid chromatography (HPLC) column is obtained from Phenomenex. The radiosynthesis is described in detail in the Supplemental Data. Following completed deprotection of [¹⁸F]3 (see Scheme 1), the crude product is purified by semi-preparative radio-HPLC using a Spherisorb ODS column (250 × 10 mm) and sodium dihydrogen phosphate buffer (70 mm, sterile) as eluent at a flow rate of 3 ml/min. [¹⁸F]1 elutes at 11–13 min and is collected directly into the final sterile product vial via a Millipore-GS 0.22 μm sterilizing filter.

The APP C99-GFP (Kaether et al, 2006 (link)) and human PS1 wild-type (hPS1WT) and the inactive hPS1D385A (hPS1DA) variant (Tamboli et al, 2008 (link)) constructs have been described previously. Filipin was obtained from Sigma-Aldrich (F9765). Primary antibodies against poly (ADP-ribose) polymerase (sc-1561; Santa Cruz Biotechnology), β-actin (A-5441; Sigma-Aldrich), liver X receptor α/β (sc-1000; Santa Cruz Biotechnology), LAMP-2 (ABL-93; Developmental Studies Hybridoma Bank), secondary horseradish-conjugated antimouse (A-9046; Sigma-Aldrich), antirabbit (A-9169; Sigma-Aldrich), and antigoat (A-5420; Sigma-Aldrich) antibodies, and secondary Alexa488-conjugated antirat (A-11006; Sigma-Aldrich) were used according to the instructions of the respective manufacturer. Standard reagents were obtained from Sigma-Aldrich unless otherwise indicated.

The SCFAs (acetate, propionate, butyrate) were measured using gas chromatography. Ten milligrams of the dried colonic digesta were mixed with 1 mL of the methanol, and shaken at 200 rpm for 90 mins at 25°C. After the mixture was centrifuged at 10,000 rpm for 10 min at 25°C, the supernatants were filtered through a 0.22 μm syringe filter, and 2 μL of the filtrates were injected into YL 6100 GC column (30 m x 0.25 mm, Youngin Chromass, Gyeonggido, Korea) to analyze SCFAs from the colonic digesta. The calibration curves were plotted using the standard reagents (Sigma-Aldrich, Saint Louis, MO, USA).

Standard reagents were obtained from Sigma-Aldrich (Vienna, Austria) or Roth GmbH & Co. KG (Karlsruhe, Germany) with the highest purity available. Restriction enzymes were obtained from Thermo Scientific (St. Leon Rot, Germany). Bacto™ peptone, Bacto™ yeast extract and Difco™ yeast nitrogen base (YNB) were obtained from Becton, Dickinson and Company (Schwechat, Austria). Zeocin™ was purchased from InvivoGen (Vienna, Austria) and linoleic acid was purchased from Sigma-Aldrich (≥ 99%, CAS: 60-33-3).

The precursor (6-OH-BTA-0) was obtained from ABX advanced biochemical compounds GmbH (Radeberg, Germany), standard reagents were obtained from Sigma-Aldrich/Merk, sterile solutions were obtained from Herlev University Hospital Pharmacy (Herlev, Denmark), and sterile Millex-GS 0.22 µm filters (SLGSV255F) were obtained from Merck Millipore. 2-butanone was dried over molecular sieves (4 Å), and the sieves were heated to 250 °C for 24 h and cooled to room temperature prior to use. [¹¹C]CH₄ was produced using an IBA Cyclone 18/18 cyclotron (Table 3) by the bombardment of the target gas, 95% N₂ + 5% H₂ (Air Products).
The automated [¹¹C]PiB radiosynthesis and product isolation was performed on a TracerMaker synthesis module (Scansys Laboratorieteknik ApS, Denmark) with a semi-preparative HPLC system with Knauer pumps (AZURA P 2.1S/P 4.1S pumps) and a Kinetex@ 2.6 μm C18 100 Å column (50 × 4.6 mm, Phenomenex). See Table 2 for details on the semi-preparative HPLC eluents and flowrates. See Section S2 (Supplementary Materials) for a detailed description of the radiosynthesis and product isolation process.

The 6-OH-BTA-0 precursor was obtained from LIMBP, Université P. Verlaine (Metz, France), standard reagents were obtained from Sigma-Aldrich/Merck (Soeborg, Denmark), and sterile solutions were obtained from Aarhus University Hospital Pharmacy (Aarhus, Denmark). Sep-Pak C18 Plus Short cartridges were obtained from Waters (Taastrup, Denmark), and sterile Millex-GV 0.22 µm filters (SLGV033RS) were obtained from Merck Millipore (Soeborg, Denmark), [¹¹C]CO₂ and [¹¹C]CH₄ were obtained by the cyclotron bombardment of the target gas, 99.5% N₂ + 0.5% O₂ (Air Liquide Danmark A/S, Taastrup, Denmark) or 95% N₂ + 5% H₂ (Air Liquide Denmark A/S, Taastrup, Denmark), respectively, either using a GE PETtrace 600, a GE PETtrace 800, or an IBA Cyclone 18/18 (see Table 3 for parameters).
Synthesis was carried out on a TracerLab FX C Pro automated synthesis module. The product was isolated by semi-preparative HPLC consisting of a Knauer P 4.1S pump fitted with a Luna C18(2) 5 µm, 250 × 10 mm column (Phenomenex, Broenshoej, Denmark). A detailed description of the [¹¹C]PiB radiosynthesis, semi-preparative HPLC, and flow charts is given in the Supplementary Materials (Section S1).