For FGF2 ELISA for EGFR mutated patients serum was collected from patients with advanced NSCLC, harboring an EGFR mutation, and treated with erlotinib at the Department of Oncology, Aarhus University Hospital (from a previously described study of patients [7] (link), and approved by the national health research committee, 1–10–72-215-17). ELISA was performed on 36 serum samples. FGF2 was quantified in duplicates of 100 μL serum with Quantikine HS ELISA kit for Human FGF basic Immunoassay (cat nr. HSFB00D, R&D, Minneapolis, MN, USA). The samples were diluted 1:2 in assay dilution buffer. The background cut-off was defined as the optical density of the blanks +3 × SD. None of the patient samples were below the cut-off.
For FGF2 ELISA for Xenograft tumor samples resected tumor tissue from the xenograft experiments was homogenized in 300–500 μL of PBS with protease inhibitors (cOmplete mini, Roche Bassel, Switzerland) and resolved in equal amounts of Cell Lysis buffer 2 (R&D, Minneapolis, MN, USA). Tissue lysates were diluted 1:20 in calibrator dilution buffer (MFB00, R&D, Minneapolis, MN, USA) prior to performing the ELISA. FGF2 was quantified in duplicates of 50 μL diluted tissue lysate with Mouse/Rat FGF basic Quantikine ELISA kit (MFB00, R&D, Minneapolis, MN, USA). The background cut-off was defined as the optical density of the blanks +3 × SD. No samples were below the cut-off.
For FGF2 ELISA for Xenograft tumor samples resected tumor tissue from the xenograft experiments was homogenized in 300–500 μL of PBS with protease inhibitors (cOmplete mini, Roche Bassel, Switzerland) and resolved in equal amounts of Cell Lysis buffer 2 (R&D, Minneapolis, MN, USA). Tissue lysates were diluted 1:20 in calibrator dilution buffer (MFB00, R&D, Minneapolis, MN, USA) prior to performing the ELISA. FGF2 was quantified in duplicates of 50 μL diluted tissue lysate with Mouse/Rat FGF basic Quantikine ELISA kit (MFB00, R&D, Minneapolis, MN, USA). The background cut-off was defined as the optical density of the blanks +3 × SD. No samples were below the cut-off.