Ephrin a5 fc

The medium on explant cultures was replaced with warm F12-MC containing recombinant human ephrin-A5-Fc, mouse EphA3-Fc (both RnD Systems, Minneapolis, MN, USA) or human Fc fragment (Calbiochem, San Diego, CA, USA). For inhibitor experiments, 30 µM Pitstop2 (Abcam, Cambridge, UK) or 400 mU/ml sphingomyelinase (Sigma-Aldrich, St. Louis, MO, USA) in F12-MC was applied during a 15 or 30 min pre-incubation, respectively, and thereafter together with ephrin-A5-Fc, EphA3-Fc or Fc. Methyl-β-cyclodextrin (2 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) was administered together with ephrin-A5-Fc, EphA3-Fc or Fc. After incubation for 20 or 120 min, cultures were fixed and stained with Alexa488 or Alexa568-phalloidin and the percentage of collapsed GCs was counted.