Ficoll paque plus media

Peripheral blood mononuclear cells (PBMCs) were obtained from fresh buffy coats and were isolated by density gradient centrifugation using Ficoll-Paque™ PLUS Media (GE Healthcare Bio-Sciences, Uppsala, Sweden). CD4⁺ T lymphocytes were enriched from PBMCs and purified by negative selection using magnetic separation according to the manufacturer´s protocol (Human CD4 T Lymphocyte Enrichment Set-DM; BD IMag™, BD Pharmingen™). The purity was evaluated by flow cytometry (>95%).

Bone marrow derived macrophages (BMDMs) were isolated and differentiated as previously described^{63 (link)}. In brief, the femurs and tibias were isolated from 8- to 12-week-old male C57BL/6 mice. After cutting off the ends of the bones and flushing with PBS, collect bone marrow using Ficoll-Paque™ PLUS Media (GE Healthcare) gradient. The macrophage precursors were differentiated in DMEM supplemented with 10% FBS, 10 ng/ml M-CSF and 1% penicillin/streptomycin at 37 °C in a humidified 5% CO₂ incubator for 7 days. For inducing alternative activation, mature BMDMs were treated with both recombinant IL-4 (20 ng/ml) and IL-13 (20 ng/ml) for 24 h and harvested for subsequent analysis.

Ascitic fluid samples were collected from 57 patients who underwent diagnostic laparoscopy or cytoreductive surgery for a suspected diagnosis of ovarian cancer in the Department of Gynecologic Oncology at The University of Texas MD Anderson Cancer Center. Forty-seven patients were diagnosed with high-grade epithelial ovarian cancer; these patient samples were included in our analysis (Table 1). All patients were enrolled in an Institutional Review Board–approved tumor banking protocol (LAB09-0890), and written informed consent was obtained to collect ascitic fluid at the time of surgery. All patients’ ascitic fluid samples were acquired only once before receiving any treatment. The collected ascitic fluid was centrifuged, and the supernatant fluid was removed; the remaining pellet was resuspended in RPMI 1640 with L-glutamine (MediaTech) and then transferred over Ficoll-Paque Plus Media (GE Healthcare) for gradient separation. The ascitic cell buffy coat was removed and washed with centrifuge steps in RPMI media. Ascitic cells, 10 × 10⁶ cells total per sample, were stored frozen in aliquots of 1 × 10⁶ cells until they were used for flow cytometry or DNA extraction prior to TCR sequencing. Clinical information and survival data were collected from medical records.

BMDM isolation and culture were performed as previously described [74 (link)]. Briefly, bone marrow was isolated from the femurs and tibias of 8- to 12-week-old male mice. The macrophage precursors purified by a Ficoll-Paque PLUS Media (17-1440-02, GE Healthcare) gradient were differentiated in DMEM supplemented with 10% FBS, 20 nM macrophage colony-stimulating factor (M-CSF) (315–02, PeproTech) and 1% P/S on bacteriological Petri dishes at 37°C in a humidified 5% CO₂ incubator. After 7 to 8 days, differentiated BMDMs were digested with 5 mM EDTA for 5 min and then plated in 6-well cell culture plates or 24-well Transwell plates in DMEM supplemented with 10% FBS and 1% P/S for further experiments.

All virus titrations and haemadsorption tests were carried out using PBMC-derived macrophages according to the protocol of the European Union Reference Laboratory for ASF in which harvesting of the PBMC by buffy coat had been replaced by separation on SepMate™ PBMC Isolation Tubes (STEMCELL Technologies, Vancouver, BC, Canada) with Ficoll-Paque™ PLUS Media (GE Healthcare, Chicago, IL, USA).

Mononuclear cells from freshly obtained WM patient’s bone marrow (BM) aspirates were isolated using Ficoll-Paque™ PLUS Media (GE Healthcare™) and treated with either ibrutinib, SYK inhibitor tamatinib or entospletinib, or combination of ibrutinib and a SYK inhibitor. Apoptosis analyses were performed on MYD88 genotyped CD19-gated lymphoplasmacytic cells (LPCs) following overnight treatment of BM mononuclear cells, as previously described^{5 (link),6 (link),23 (link)}. Subject participation was approved by the Harvard Cancer Center/Dana-Farber Cancer Institute Institutional Review Board, and all participants provided written consent for sample use.