An In Vitro Technique for UV Irradiation-Mediated Generation of Apoptotic T Cells
Transcrição
Begin by culturing Jurkat T cells in basal cell culture medium, supplemented according to the manuscript directions, at 37 degrees Celsius and 5% carbon dioxide. Jurkat T cells are a suspension cell line that can be maintained through passaging into pre-warmed culturing media every three days.
Prepare apoptotic cells by growing them to 90% confluency, which takes three to four days after passaging. Grow cells in five T75 flasks, to obtain a sufficient number of cells for this protocol. Once the cells are ready, aspirate the flask contents about 24 milliliters, and transfer them to a 50-milliliter conical tube. Count the cells using trypan blue staining and a hemocytometer.
Pellet the cells by centrifugation at 271 times g for five minutes, and discard the supernatant. Then, resuspend the cells in media to obtain three million cells per milliliter. Aliquot the cells into 100-by-20 millimeter tissue culture dishes by transferring five milliliters of cells per dish.
Prepare approximately nine culture dishes. Set one dish aside as an unexposed control, and expose the remaining dishes to UV. Set the UV crosslinker to the correct energy level by pressing the energy button and entering "600" with the number pad. Irradiate all dishes with the cells except for the control, making sure to remove the top cover of the tissue culture dish during UV exposure.
Then, incubate all the dishes in a cell culture incubator at 37 degrees Celsius and 5% carbon dioxide for four hours. After the incubation, combine all the irradiated cells into a 50-milliliter conical tube, and pellet them by centrifugation at 271 times g for five minutes. Discard the supernatant, and resuspend the cells in 24 milliliters of sterile PBS. Centrifuge the tube again, and remove the supernatant.
