In order to measure the migration of the EPCs, 1.5×105 cells at passage 4, 6 and 8 with or without pre-treatment with tyrphostin AG1295 (Sigma-Aldrich) at 20 µM for 1 h were seeded in the upper Transwell chamber (BD Biosciences) in serum-free medium, with 500 µl DMEM with 10% FBS in the lower chamber. After 24 h, cells that did not migrate through the pores were carefully wiped out with a cotton-tipped swab. The filters were fixed in 90% alcohol, followed by staining with 0.1% crystal violet (Meryer, Shanghai, China). After washing with PBS 3 times, the filters were observed under an inverted microscope (Olympus).
Tyrphostin ag1295
Manufactured by Merck Group
Tyrphostin AG1295 is a tyrosine kinase inhibitor. It functions as a selective inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase.
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2 protocols using tyrphostin ag1295
1
Transwell Assay for EPC Migration
2
PDGFR-β Modulation of EPC Function
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To examine the effects of PDGFR-β, EPCs at passage 4, 6 and 8 were pre-treated with PDGF-BB (PeproTech, Rocky Hill, NJ, USA) at 40 ng/ml for 24 h. The cells were then used in the subsequent experiments. To examine whether the PDGFR-β/PI3K signaling pathway is involved in the PDGF-BB-induced biological function changes of EPCs, EPCs were treated with 20 mM tyrphostin AG1295 (Sigma-Aldrich) for 1 h, followed by PDGF-BB stimulation as mentioned above. The cells were then used in the subsequent experiments.
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