Chrompure human igg

NCI-N87 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and OE-19 cells were obtained from the European Collection of Cell Culture (ECACC, Porton Down, UK). The cell culture medium was RPMI-1640 supplemented with 10% fetal bovine serum (FBS), and antibiotics and cells were cultured at 37°C under 5% CO₂. Trastuzumab and palivizumab was produced by Genentech (South San Francisco, CA, USA) and MedImmune, LLC (Gaithersburg, MD, USA), respectively. ChromPure human IgG (Jackson ImmunoResearch, West Grove, PA, USA) was used as human IgG control antibody for in vitro assays. IgG antibodies were produced using the Freestyle 293 system (Invitrogen, Carlsbad, CA, USA) and purified using protein-A affinity chromatography (GE Healthcare, Piscataway, NJ, USA). Endotoxin was removed with an Endotoxin Removal Kit (GenScript, Piscataway, NJ, USA), and endotoxin levels were determined using an Endotoxin Detection Kit (GenScript). Recombinant proteins were produced as secreted proteins using the Freestyle 293 system and purified using protein-A or Ni-NTA chromatography (Qiagen, Valencia, CA, USA) for Fc-tagged or His-tagged proteins, respectively.

Activation of Notch signalling in the cell lines used to select the signature was achieved as previously described [47 (link)] with minor modifications: 1µg rat Jagged1-Fc (cat #599-JG, R and D systems) or ChromPure Human IgG, Fc (cat #009-000-008, Jackson ImmunoResearch) was used instead of 2 µg. Activation of Notch signalling in the cell lines used to establish a Notch transcriptomic signature was performed as described above with minor modifications: Tissue culture-treated plates were incubated over night at 4 °C or for 1 h at room temperature with 5-µg/ml Protein G (cat #21193, Thermo Fisher Scientific) in PBS. Non-specific binding was blocked by incubating coated plates with 1 × blocking buffer (Thermo Fisher Scientific, cat #37525), and plates were incubated with immobilized ligand or Fc control for 1.5 h at room temperature. Blockage of Notch signalling was in both cases achieved by adding 10-µM DAPT (InSolution™ γ-Secretase Inhibitor IX, cat #565784, MerckMillipore) directly to the cell culture medium, and cells were incubated with DAPT for 8 h and 72 h. Non-GSI-treated cells (Fc control, Jagged1-Fc) were treated with equimolar volumes of carrier control (DMSO, cat #D8418-50ML, Sigma-Aldrich).