Granulocyte macrophage colony stimulating factor gm csf

Bone marrow aspirates or peripheral blood samples were obtained from AML patients. Normal peripheral blood samples were obtained from healthy donors and cord blood samples were obtained from the freshly delivered placenta of the healthy donor after informed consent using protocols approved by the Penn State College of Medicine Institutional Review Board (IRB). Mononuclear cells (MNCs) were isolated by density gradient separation (Ficol-Paque, GE Healthcare Life Sciences, Pittsburgh, PA) and frozen for later use. Primary human AML and normal cells were cultured in the serum-free expansion medium (SFEM) (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with recombinant human stem cell factor (SCF), Interleukin 3 (IL-3), FMS-like tyrosine kinase ligand (FLT3-L), Granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). All cytokines were purchased from Shenandoah Biotechnology, Warwick, PA. StemRegenin1 (SR1) (ChemieTek, Indianapolis, IN), and UM729 (Selleckchem, Houston, TX) were added to maintain the “stemness” of primary cells for stem cell assays (25 (link)). All cultures were supplemented with 1% Penicillin and Streptomycin and stored in a humidified 37°C incubator with 5% CO₂.

Monocyte isolation and DC differentiation were performed as described previously.⁴ (link) Briefly, monocytes were obtained using the adherence method, where PBMCs were plated in adherent plastic plates (Sarstedt, Nümbrecht, Germany) in medium (X-Vivo 15 medium, Lonza, Basel, Switzerland) supplemented with 5% human serum, 2 mM L-glutamine and 1 mM sodium pyruvate (Thermo Fisher Scientific, Waltham, MA, USA), 1,000 U/mL (100 ng/mL) IL-4, and 800 U/mL (50 ng/mL) Granulocyte-macrophage colony-stimulating factor (GM-CSF) (both from STEMCELL Technologies) and incubated in a CO₂, 37°C incubator for 7 days. On day 4, the medium was replaced with fresh medium supplemented with IL-4 and GM-CSF. On day 7, DCs were matured with maturation medium containing 1,000 U/mL (100 ng/mL) IL-4, 800 U/mL (50 ng/mL) GM-CSF, 10 ng/mL tumor necrosis factor alpha (TNF-α) (STEMCELL Technologies), 1 μg/mL PGE2 (Sigma), 10 ng/mL IL-1β (Feldan, Quebec City, QC, Canada), and 100 ng/mL IL-6 (Miltenyi Biotec, Bergisch Gladbach, Germany) and loaded with the desired peptide (1 μg/mL LMP2_426–434, [CLGGLLTMV] or 1 μg/mL WT1_37–45 [VLDFAPPGA], both from JPT Peptides (Berlin, Germany). Last, DC medium was supplemented with interferon γ (IFN-γ; 1,000U/mL, Feldan) for the last 24 h of maturation.

Peripheral blood mononuclear cells (PBMCs) were isolated from freshly drawn venous blood by Lymphoprep (StemCell, Vancouver, British Columbia, Canada) centrifugation. The PBMC monocytes were achieved by adherence in Monocyte Attachment Medium (Promocell, Heidelberg, Germany) for 2 h. Monocyte derived dendritic cells (moDCs) were cultured in X-Vivo 20 medium (Lonza, Basel, Switzerland) supplemented with 40 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) (StemCell) for 8 days and 20 ng/mL IL-4 (PromoKine, Heidelberg, Germany) for 5 days and 50 ng/mL IL-1β (StemCell) and 50 ng/mL TNF-α (Biotechne, R&D Systems, Minneapolis, MN, USA) in the 6th day of culture. Monocyte derived macrophages (moMφs) were specialized from PBMC monocytes by stimulation with 20 ng/mL macrophage colony-stimulating factor (M-CSF) (StemCell) for 10 days in Macrophage Base Medium DXF (PromoCell).