This protocol is easy to perform. The cloning phase allows a larger number of T clones to be obtained from fresh samples. Moreover, the phenotype and function of T cells can easily be analyzed.
Demonstrating the procedure will be Mary Christopher, a doctorate student from my laboratory. To induce T-cell cloning, use the bag spike with the needle-free valve to open an irradiated buffy coat bag in a laminar flow hood. Transfer up to 50 milliliters of blood from the bag into a T-150 flask, and add 70 milliliters of PBS to the flask.
Carefully layer 30 milliliters of the diluted blood onto 20 milliliters of density gradient in each of four 50 milliliter conical tubes, and separate the cells by density gradient centrifugation. Carefully aspirate the supernatant, and transfer the PBMC from each tube into a single new 50 milliliter tube. Use PBS to bring the final volume to 50 milliliters and collect the PBMC by centrifugation.
After a second wash under the same conditions, suspend the isolated feeder cells in 10 milliliters of cell culture medium for counting, and add 25 milliliters of a 70 milliliter cell culture medium supplemented with 50 units of IL-2 solution into each of two 50 milliliter tubes. Then add feeder cells to one tube at a 2 by 10 to the 6th cells per milliliter of cell culture medium supplemented with IL-2 concentration. To set up a T lymphocyte culture, pool the T cell clone containing supernatants from stenotic valve sample flask into a single 50 milliliter tube, and collect the cells by centrifugation.
Wash the pellet three times in 50 milliliters of PBS per wash, before resuspending the cells in 1 milliliter of cell culture medium for counting. Dilute the cells to a 1 by 10 to the 3rd cells per milliliter concentration, and add 500 microliters of cells to the tube of cell culture medium supplemented with IL-2 without feeder cells. Add the feeder cell suspension to a plastic 100 by 15 milliliter Petri dish, and add 100 microliters of feeder cells to each well of the appropriate number of U-bottom 96-well plates for the experiment.
Then add the T-cells to a Petri dish, and add 100 microliters of T-cells to each feeder cell supplemented well for a one week incubation in the cell culture incubator. The PBMC isolation is vital for obtaining feeder cells and enabling the detection and characterization of infiltrating leukocytes in aortic valve samples. After two weeks of incubation, a clone T-cell population can be obtained.
Here, the gating scheme for analyzing T-cell subpopulations in patients with calcific aortic valve disease is shown. As observed in this patient, there were more CD4+T-cells than CD8+T-cells. Flow cytometric analysis of uncloned T-cells confirms that the same T-cell markers are present in both native valve and clone samples.
Taken together, these data indicate that T-cells are present in calcified aortic valves with CD45+leukocytes, suggesting that calcific aortic valve disease is linked with activation of the immune system and inflammatory activity. This method is very versatile. It can be applied to different protocols for which researcher need to be able to isolate cells from a tissue, for the functional characterization.
Keep in mind that it is important to work under sterile conditions to avoid contamination, and to critically observe the cells under the microscope.
