Effect of prostacyclin in ovarian carcinoma
Effect of prostacyclin in ovarian carcinoma
Protocol ascTAM isolation: • Ascites were collected from patients with ovarian carcinoma undergoing primary surgery at the University Hospital in Marburg. • approval of collection and analysis of human material by the ethics committee at Philipps University (reference number 205/10) • ascTAM were isolated from ascites by a combination of density gradient centrifugation, filtration using 30μm and 100μm cell strainer, followed by magnetic cell sorting (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) • Cell populations with a purity of >95% CD45+ cells, as determined by flow cytometry, were cryopreserved for subsequent functional assays, Protocol CAF isolation and culture:• omental tissue with metastasis was collected from patients with ovarian carcinoma undergoing primary surgery at the University Hospital in Marburg • Omental CAF were separated from omental tumor tissue by trypsin digestion (2 h at 37°C) with following treatment with a mixture of 18.5 U/ml collagenase and 2.5 μg/ml hyaluronidase (Sigma Aldrich) in fibroblast culture medium (DMEM/HAMs F12 (1:1), 10 % FCS, 10 ng/ml EGF, 1 % Pen/Strep) overnight at 37°C • disgested tissue was filtered through a 100 μm mesh and washed • CAF enriched fraction was precultured in fibroblast culture medium • if necessary MACS depletion of CD45+ and EpCAM+ cells / positive selection via anti- fibroblast beads (Miltenyi Biotec) was performed • for functional assays pure CAF were cultured for a maximum of three passages in OCMI/50%Ascites. Protocol: Flow cytometric analysis of cell phenotypes: • Characterization of cells (ascTAM, CAF, M1-MDM, asc-MDM) by flow cytometry on a FACS Canto II instrument using Diva Software (BD Biosciences) and analysis by FlowJo™ v10.8 Software (BD Life Sciences) • ascTAM and MDM were identified with anti-human CD14-FITC (Miltenyi Biotech) • CAF were identified by negative staining with anti-EpCAM-Vioblue and anti-CD45-FITC • further discrimination of CAF: via fibroblast markers anti-human CD140a-PE (eBioscience/Thermo Fisher Scientific) and FAP-PE (R&D Systems/Thermo Fisher Scientific) in combination with anti-human mesothelin-APC (R&D Systems) and intracellular staining with anti-human cytokeratin-APC and anti-human imentin-FITC (both from Miltenyi Biotec). M1-MDM cells are differentiated in vitro.  Protocol: Differentiation of monocyte-derived macrophages (MDM) from healthy donors • Leucoreduction system chambers from healthy adult volunteers, provided by the Center for Transfusion Medicine and Hemotherapy at the University Hospital Gießen and Marburg • Monocytes were isolated by Ficoll density gradient centrifugation and following purification by adherence selection or via CD14+ MACS microbeads • Differentiation of monocytes: ◦ Approximately 3x106 monocytes per 6-well ◦ culture for 7 days prior to treatment and RNA collection ◦ TAM-like asc-MDM: in cell-free ascites pool derived from 10 patients ◦ m1-MDM: differentiation in RPMI1640 (Life Technologies, Darmstadt, Germany) supplemented with 5% human AB serum (Sigma Aldrich), 1% sodium pyruvate (Sigma Aldrich), and 100 ng/ml granulocyte macrophage colony-stimulating factor (CSF) (Peprotech, Hamburg, Germany) for 5 days followed by activation with 100 ng/ml LPS (Sigma Aldrich) and 20 ng/ml IFNγ (Biozol, Echingen, Germany) for 2 days PBMCs were thawed and cultivated for 7 days in RPMI (5 %AB-Serum, 1 % Sodium-Pyruvat. 100 ng/mL GM-CSF were added on day 0 (until day 7), 100 ng/mL LPS and 20 ng/mL IFNj were added on day 5 (until day 7). Half renew of media was performed on day 5. Format: 6 well, 4 x 10^6 cells  On day 7 media was changed to serum free media (RPMI/1 % So.-Py.)for 24 h. cells were treated with 1 μM MRE for 5 h. RNA was isolated with NucleoSpin® RNA II kit purchased from Macherey Nagel. RNA was eluted in 30 μL water.  DNAse digestion was performed with DNA-freeTM DNA Removal kit purchased from Thermo Fischer Scientifi Lexogen Quantseq