Ficoll paque premium solution

Immunodeficient NSG (NOD/LtSz-scid/IL2Rγnull) mice were purchased from Jackson labs (catalog number: 005557). Mononuclear cells (CBMCs) were isolated from fresh umbilical human cord blood (Umbilical Cord Blood Collection Program, UC Davis Health System) using Ficoll-Paque Premium solution (GE healthcare). CBMCs were infected with either P3HR1, B95.8 or AG876 viruses in vitro for 1.5 hours at 37°C. A minimum of 10 million cells were injected intraperitoneally into 6- to 10- week-old NSG mice which were age-matched. Mice were kept for a maximum of 92 days after injection of cord blood or were sacrificed if they become moribund prior to day 92. P3HR1 studies were performed using four different donors. In general, mice received 20,000 infectious units of the AG876 and B95.8 viruses (a dose sufficient to induce tumors in essentially 100% of infected animals [43 (link),47 (link)]. Animals infected with the P3HR1 virus received 500,000 to 1 million infectious units; lower amounts of virus were not examined due to the expense of the model (and no difference was observed in the ability of different virus doses to cause tumors). P3HR1 was not tittered in three of the infected mice (S1 Table).

Bone marrow tissues were isolated from the rats’ tibia and femoral bones according to previously described procedures [3 (link)]. Briefly, animals were euthanized by intraperitoneal injection of ketamine (100 mg/mL) and xylazine (100 mg/mL) (both from Ilium Troy Laboratory, Blacktown, Australia). The central canal of the bone was flushed with 2 mL of Dulbecco Modified Eagle’s Media (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (pen/strep) (all from Gibco, Life Technologies, Carlsbad, CA, USA) to extrude the marrow tissue. Mononuclear cells were purified using Ficoll-Paque PREMIUM solution (GE Healthcare Bioscience, Uppsala, Sweden) and the extracted cells were cultured on T25 cm² flasks in a 37 °C humidified chamber with 5% CO₂. After 24 h, the non-adherent cells were removed and the attached cells were allowed to grow. Complete media for BMSC proliferation (DMEM + 20% FBS + 1% pen/strep + 1% nonessential amino acids) was used to maintain the cells. The cells were subcultured until passage 3 (P3) and were then characterized by Fluorescein isothicyanate (FITC)-conjugated CD90 mouse monoclonal antibody (1:200; Thermo Scientific, Waltham, MA, USA), a surface marker for BMSCs. Translineage differentiation was conducted using fourth generation (P4) of cells.

Human peripheral blood was collected in EDTA tubes (BD Vacutainer) from healthy volunteers enrolled in University of Maryland Institutional Review Board (IRB) approved protocol HP-40025-CVD4000, and methods were conducted in compliance with approved Environmental Health and Safety guidelines (IBC 00003017). PMN were obtained by Ficoll-Paque (Premium solution; GE Healthcare Bio-Sciences AB, Sweden) gradient centrifugation following dextran (Alfa Aesar, USA) sedimentation (74 (link)). Contaminating erythrocytes were removed by hypotonic lysis. After being washed, cells were suspended in enteroid differentiation medium (DFM) without antibiotics and immediately used. The cell suspension contained >95% PMN, as determined by flow cytometry and May-Grünwald-Giemsa-stained cytopreparations. PMN viability was >98%. Cell counts were determined using the Guava ViaCount reagent (Luminex, USA); viable and nonviable cells were distinguished based on differential permeabilities to two DNA-binding dyes. Cells were stained following the manufacturer’s instructions and analyzed in Guava 8HT using ViaCount software (Luminex, USA).