Animal free recombinant human egf

The following GSC culture medium was applied to enrich GSCs: DMEM/F12 medium (Thermo Fisher Scientific, Grand Island, NY) supplemented with 1 × B-27™ Supplement, serum free (Gibco, 17504044), 20 ng/ml Animal-Free Recombinant Human EGF (PeproTech, AF-100-15), 20 ng/ml Recombinant Human FGF-basic (154 a.a.) (PeproTech, 100-18B), and 1× penicillin–streptomycin. Cells were seeded in Costar^® 24 Well Clear Flat Bottom Ultra Low Attachment plates (Corning, NY) at 1000 cells/ml. Spheres were carefully aspired into 15 ml tube, and spin down at 1000 rpm for 3 min. Subsequently, 1 ml warm trypsin was used to digest spheres at room temperature for 3 min. The reaction was stopped by adding 10 ml GSC culture media, then single cells were spin down and re-plated.

Adherent tumour cells were harvested using trypsinization, and single cells were resuspended with DMEM/F12 (DMEM/F12, BI, Haemek, Israel) medium containing 4 μg/mL heparin (Heparin sodium salt, Sigma‐Aldrich), B27 (cat. no. C11330500BT, B‐27 Supplement (50×), serum free, Gibco), 20 ng/mL human recombinant epidermal growth factor (cat. no. AF‐100‐15, Animal‐Free Recombinant Human EGF, PeproTech) and 20 ng/mL human recombinant basic fibroblast growth factor (cat. no. 100‐18B, Recombinant Human FGF‐basic, PeproTech) after centrifugation. Single cell suspensions were grown on 6‐well ultralow attachment plates (ultralow attachment plates, Corning) at a concentration of 3000 cells/mL at 37°C for 10‐14 days. Then, the spheres were photographed using a microscope and counted with ImageJ cell counter.

Animal‐Free Recombinant Human EGF was obtained from PreProtech (London, UK). Stock solutions were prepared in water and stored at −80 °C. Canertinib (CI‐1033, Selleckchem, Houston, TX, USA), U0126 (Selleckchem) and LY294002 (Calbiochem, Darmstadt, Germany) were prepared in DMSO and stored at −20 °C. pTH‐related protein (1–34) amide trifluoroacetate salt [PTHLH (1–34), Bachem, Switzerland] was prepared in 0.1% BSA, 1 mm HCl at 250 μm and stored at −80 °C.