Kp 10

The in vitro migration assay was performed in the transwell inserts, with an 8-μm pore size, the membrane of which was uncoated (SPL, Germany). The cells were treated by PBS (Phosphate buffered saline) with 10-100 or 500 nM kp-10 (Anaspec, USA) for 24 hours and one other group of cells was not treated with kp-10. Then the cells were trypsinized and a number of 2 × 10⁴ mesenchymal stem cells in 200 μl serum-free medium were seeded into the top chamber. Then, 30% of Fetal calf serum (FCS) or 4 × 10⁴ melanoma cells in 500 μl medium were added to the lower chamber as a chemoattractant. After 24 hours of incubation at 37˚C, the non-migrated cells were removed from the top of the membrane, whereas, cells on the bottom of the membrane were trypsinized with 300 μl of trypsin and were suspended into the medium and counted in five minutes by flow cytometry (BD Calibur^™ (Becton Dickinson)) after obtaining the appropriate gate. Results were obtained by three experiments.[23 (link)24 (link)]

Expression of the CXCR4 protein was determined in the MSCs of the fourth passage. A number of 5 × 10⁴ mesenchymal stem cells, after 24 hours of treatment with 10-100 or 500 nM kp-10 (Anaspec, USA), and also no treated cells, were trypsinized. Then the cells were washed with PBS twice and incubated with 5 μl of anti-mouse CXCR4 PE (dilution 1:200, eBioscience, USA) or Rat IgG isotype control antibody for 45 minutes in the dark, under room temperature, and analyzed with flow cytometry (BD Calibur^™ (Becton Dickinson)). The results were indicative of three experiments.[24 (link)27 (link)]