Recombinant mouse hgf protein

Unless otherwise stated, all chemicals including mouse monoclonal anti-β-actin were obtained from Sigma-Aldrich Chemical Co., St. Louis, MO, U.S.A. The recombinant mouse HGF protein was obtained from R&D Systems, Minneapolis, MN, U.S.A.; rabbit polyclonal anti-mouse Fpn1 from Novus Biologicals, Littleton, CO, U.S.A.; mouse anti-human TfR1 from Invitrogen Life technologies, Carlsbad, CA, U.S.A.; rabbit polyclonal anti-ferritin light chain (Ft-L) and anti-DMT1 from Proteintech, Chicago, IL, U.S.A.; and rabbit monoclonal anti-IRP1 and anti-IRP2 were from Abcam, San Francisco, CA, U.S.A. Goat anti-rabbit or anti-mouse IRDye 800 CW secondary antibodies were bought from LI-COR Bio Sciences, Lincoln, NE, U.S.A.; the bicinchoninic acid (BCA) protein assay kit and AevertAid First Strand cDNA Synthesis Kit from Thermo Fisher Scientific, Waltham, MA, U.S.A.; the TRIzol reagent from Life technologies, Carlsbad, CA, U.S.A.; FastStart Universal SYBR Green Master and LightCycler96 from Roche, Nutley, NJ, U.S.A.; protein RIPA lysis buffer from Beyotime Institute of Biotechnology, Haimen, JS, China. Recombinant HGF protein was dissolved in phosphate buffer saline (PBS) and diluted to 1–120 ng/ml before use.

The next day, cells were transferred from PMEA chamber slide to Matrigel-coated culture plates. Cells in the chamber slide were washed twice with PBS, and then overlaid with Gibco TrypLE^TM Express (Thermo Fisher Scientific), and incubated at 37 °C for 10 min. Cells were collected in standard medium supplemented with 10% FBS, and then centrifuged at 300× g for 5 min. After discarding the supernatant, cells were recovered with modified ES medium [48 (link)] and seeded on Matrigel (Corning, NY, USA)-coated 24- or 96-well plates. Fresh ES medium was added every 2 or 3 days.
The modified ES medium contained Advanced DMEM/F12 (Thermo Fisher Scientific), Penicillin-Streptomycin Solution Hybri-Max^TM (100 ng/mL; Sigma Aldrich, MO, USA), gentamycin sulfate solution (25 mg/mL, Wako Pure Chemical, Osaka, Japan), HEPES buffer solution (10 mM; Thermo Fisher Scientific), B-27 supplement (Thermo Fisher Scientific), bovine serum albumin (1 mg/mL; Sigma Aldrich), Glutamax (Thermo Fisher Scientific), recombinant murine Noggin (50 ng/mL; Pepro Tech, Cranbury, NJ, USA), recombinant human R-Spondin1 (500 ng/mL; R&D Systems, Minneapolis, MN, USA), recombinant murine EGF (50 ng/mL; Pepro Tech), recombinant mouse HGF protein (50 ng/mL; R&D Systems), recombinant mouse Wnt-3a protein (100 ng/mL; R&D Systems), Culture Sure Y-27632 (10 μM; Wako), and hydrocortisone (0.5 μM).