Cfi apo tirf 100 h 1

Keratocytes of Central American cichlids (Hypsophrys nicaraguensis) were cultured in culture medium (Leibovitz’s medium, L-15, L5520, Sigma-Aldrich, St Louis, MO) supplemented with 10% FBS (Nichirei, Tokyo, Japan) and antibiotic/antimycotic solution (09366-44, Nacalai Tesque, Kyoto, Japan) as previously described (16 (link)). All methods were carried out in accordance with national guidelines and the Regulation on Animal Experimentation at Yamaguchi University. All experimental protocols were approved by Yamaguchi University Animal Use Committee. Cells were treated with trypsin (0.5 g/liter) and 0.53 mM EDTA (trypsin-EDTA, 32778-34, Nacalai Tesque) for 30 to 60 s to separate any cell-cell adhesions. The single keratocytes ware treated with the culture medium containing 250 nM HMRef for 10 min. Then, the medium was replaced with the culture medium containing no probe. The migrating keratocytes were observed using an inverted microscope (Ti; Nikon, Tokyo, Japan) equipped with a laser confocal scanner unit (CSU-X1; Yokogawa, Tokyo, Japan) with a 100× objective lens (CFI Apo TIRF 100×H/1.49, Nikon, Tokyo, Japan). The fluorescence images were detected using an electron multiplying CCD camera (DU897, Andor, Belfast, UK).