87 TSPO in neurodegeneration according to Haenseler et al 201761. In short, on day 0 iPSCs were dissociated with TrypLE Express (Gibco) and 4x10^6 iPSCs were added to one well of 24-well AggreWellTM 800 (Stem Cell Technology) according to the manufacturer’s protocol in 2ml EB medium (E8, SCF (20ng/ml, Miltenyi), BMP4 (50ng/ml; Gibco), VEGF (50ng/ml, PeproTech)) with 10uM ROCK inhibitor (Y-27632, Abcam). From day 1 to 6, 75% medium was exchanged with fresh EB. On day 7 embryoid bodies were transferred to 2x T175 flasks containing factory medium (XVIVO-15 (Lonza), 2mM GlutaMAX, 50uM 2-Mercaptoethanol, 25ng/ml IL-3, and 100ng/ ml M-CSF (all Gibco)) and fed weekly with factory medium. Starting from week 4 after transfer, medium was removed and tested for the presence of primitive macrophages using CD45 (immunotools), CD14 (immunotools) and CD11b (Biolegend) immunopositivity by flow cytometry (FACSCalibur, BD Biosciences). Primitive macrophages were transferred to microglia medium (SILAC Adv DMEM/F12 (Gibco), 10 mM glucose (Sigma), 2 mM GlutaMAX, 0.5 mM L-lysine (Sigma), 0.5 mM L-arginine (Sigma), 0.00075% phenol red (Sigma), 100ng/ ml IL-34 (PeproTech), 10gn/ml GM-CSF (Gibco)), fed every 3-4 days and used for experiments after 7 days. Drug treatments and cell activation Cells were treated with XBD-173 at the indicated concentrations for 1h prior to LPS activation or for 20h prior to phagocytosis. Pro-inflammatory activation was induced with lipopolysaccharide (100ng/ml; Sigma) for 24h. For live-cell phagocytosis assays, pHrodo®- labelled zymosan A bioparticles (Thermo) were added to the culture medium and incubated for 2h at 37°C with 5% CO2. pHrodo®-fluorescence intensity was acquired in a plate reader (Cytation5, BioTek) or by Flow cytometry (FACSCalibur, BD Biosciences). Cytokine analysis Cytokines were assessed from cell-free cell culture supernatant using enzyme-linked immunosorbent assay (ELISA) according to the manufacturers’ protocols. The following assays were used: mouse-TNFα and mouse-IL-6 ELISA (R&D Systems), huma-TNFα and human-IL-6 (BD Biosciences). Absorbance was measured in a Spark plate reader (Tecan). RNA sequencing RNA was extracted from control and LPS treated (100ng/mL, 24 hours) primary human macrophages using the RNeasy Mini Kit. cDNA libraries (Total RNA with rRNA depletion) were prepared and sequenced using a HiSeq4000. Lanes were run as 75 bases Paired End. Sequencing depth was minimum 40 million reads per sample. LC-MSMS analysis of supernatant for XBD173 concentration Supernatant samples were stored at -20°C or lower until analysis. Samples (25 µL) were prepared for analysis by protein precipitation with acetonitrile containing internal standard (tolbutamide) (200 µL) followed by mixing (150 rpm, 15 min) and centrifugation (3000 rpm, 15 min). The supernatant (50) µL was diluted with water (100 µL) and mixed (100 rpm, 15min). Samples were analysed by LC-MSMS (Shimadzu Nexera X2 UHPLC/Shimadzu LCMS 8060) with Phenomenex Kinetex Biphenyl (50 x 2.1)mm, 1.7 µm column and mobile phase components water/0.1% formic acid (A) and acetonitrile/0.1% formic acid (B). Mobile phase gradient was 0 to 0.3 min 2% B; 0.3 to 1.1 min increase to 95% B; 1.1 to 1.75 min 95% B, 1.75 to 1.8 min decrease to 2% B; 1.8 to 2.5 min 2% B. Flow rate was 0.4 mL/min. Injection volume was 1 µL. Calibration standards were prepared by spiking XDB173 into control supernatant
RkJQdWJsaXNoZXIy MTk4NDMw