To begin, isolate the resident vascular wall CD34-positive stem cells from murine models. Obtain pure cells by magnetic activated cell sorting and characterize them. For detecting calcium signaling, obtain a coverslip pre-seeded with the cells and wash it once with PBS.
Then incubate the cells with Fura 2-AM and Tyrode solution for 30 minutes in the dark. To remove the dye, incubate the cells with Tyrode solution for 10 minutes. Then mount the chamber on the stage of an inverted fluorescence microscope, open the main window and go to the grab settings dialogue and camera page.
Press the live button for the live image display. To acquire images, draw several freehand lines to circle the cells with different colors and predefine the regions of interest. Fluorescence microscopy demonstrated that ATP administration increased the intracellular calcium levels in the isolated vascular wall CD34-positive stem cells.
