To characterize the CD34+stem cells isolated from the murine aorta and mesenteric arteries, perform tissue block culture and obtain the cells by magnetic separation. To detect the endothelial cell in fibroblast cell marker expression after differentiation, observe the cells at 40x magnification with laser excitations of 488 nanometers. After trypsinizing the cells, pellet them by centrifugation and resuspend in 100 microliters of sorting buffer.
Then incubate them with anti-mouse CD16/CD32 monoclonal antibodies at four degrees Celsius for five minutes. Then add two microliters of each desired monoclonal antibody for immunofluorescent staining and incubate again at 4 degrees Celsius for 10 minutes. Wash the cells twice with one milliliter of buffer.
After centrifuging the cells as demonstrated, remove the supernatant and resuspend the cells in 300 microliters of buffer. Transfer the cells to the flow tubes and place them in an ice box. Run flow cytometry to check the percentage of differentiated cells.
Flow cytometric evaluation confirmed the high purity of isolated CD34+vascular wall stem cells. Cellular immunofluorescent staining showed that the isolated CD34+stem cells predominantly expressed stem cell markers, CD34, c-kit, and Flk-1. Flow cytometry also confirmed that the isolated CD34+stem cells possessed the ability to differentiate into endothelial cells as well as fibroblasts in vitro.
