Resident of vascular wall CD34 positives in stem cells are increasingly recognized for their crucial role in regulating vascular remodeling post-injury. Establishing a stable and efficient method to character functional CD34 positive cells is essential for further investigating the participating mechanisms under various physiological and pathological conditions. This technique combines magnetic B-screening and fluid cytometry to purify primary cultured resident CD34 positive stem cells.
Then the purified cells can be functionally identified through immunofluorescent stain and calcium imaging. Due to the phenotypical and functional heterogeneity of resident CD34 predictive stem cells in the vascular wall, it will decelerate to identify the specific subcellular type of relevant muscular CD34 proactive cells. We present a refined methodology for the culture identification and the functional assessment of vascular wall-resident CD34 positive stem cells.
This novel approach encompasses magnetic sorting photocytometry immuno fluorine sterling, and custom measurement. By employing these techniques, our study is so only the foundation for the future comprehensive investigations into the function under regulation of reticent CD34 positive stem cells under both physiological and pathological conditions. To begin, placing an anesthetized mouse on the operating table.
Using sterile ophthalmic scissors and forceps, open the thoracic and abdominal cavities. This will expose the heart and the intestines. Carefully dissect the mesenteric arteries and the aorta separately.
Rinse the mesenteric arteries and PBS and transfer them to a 10 centimeter Petri dish containing 1%penicillin streptomycin amphotericin B solution. Similarly, rinse the aorta and transfer it to another Petri dish containing the same solution. Quickly remove all the fat around the aorta and the mesenteric arteries.
After rinsing twice, place the arteries under a stereo microscope in a tissue culture hood. Carefully make a longitudinal cut along the arteries. Then using fine forceps, peel off the outer layer of the aorta and the first branch of the mesenteric arteries.
Place the outer layer in a 3.5 centimeter Petri dish containing 0.1 to 0.2 milliliters of culture medium, and chop it into tiny pieces of about one cubic millimeter. Transfer the tissue pieces into a gelatin coated T25 flask. Place the flask vertically in an incubator at 37 degrees Celsius with 5%carbon dioxide.
After three hours, add five milliliters of DMEM high glucose growth medium to the flask to submerge all the tissue blocks. To begin, isolate and culture the outer layer of the aorta and the first branch of the mesenteric arteries isolated from the anesthetized mouse. Once the cells become 80%confluent, centrifuge the dissociated cell suspension at 300 G for five minutes at room temperature.
Discard the supernatant completely and re-suspend the cell pellet in 98 microliters of sorting buffer per 10 million total cells. After the addition of CD34 antibody, incubate the cells in the dark for 30 minutes at four degrees Celsius. To wash the cells, add two milliliters of sorting buffer to the cells and centrifuge at 300 G for 10 minutes.
After discarding the supernatant, resuspend the cells in 80 microliters of sorting buffer. Then add 20 microliters of Anti-FITC MicroBeads to the cell suspension. After washing and centrifuging the cells as demonstrated previously, resuspend the cells in one milliliter of sorting buffer.
Next, position a magnetic separation column within the magnetic field of a separator, and place a collection tube beneath the column. After rinsing with 500 microliters of buffer, load the cell suspension into the column and collect the flow through containing unlabeled cells in the collection tube. Remove the column from the separator and place it on a fresh 15 milliliter centrifuge tube.
Introduce 500 microliters of sorting buffer into the column and push the plunger into the column to flush out the magnetically labeled cells. To characterize the CD34 positive stem cells isolated from the murine aortal and mesenteric arteries, perform tissue block culture and obtain the cells by magnetic separation. To detect the endothelial cell and fibroblasts 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, CD 16, CD 32 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 four 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 icebox.
Run flow cytometry to check the percentage of differentiated cells. Flow cytometric evaluation confirmed the high purity of isolated CD34 positive vascular wall stem cells. Cellular immunofluorescent staining showed that the isolated CD34 positive stem cells predominantly expressed stem cell markers, CD34, c-kit and Flk-1.
Flow cytometry also confirmed that the isolated CD34 positive stem cells possessed the ability to differentiate into endothelial cells as well as fibroblasts in vitro. 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 cover slip preceded 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.
