Analysis of Efferocytosis of Apoptotic Thymocytes by Peritoneal Macrophages

To harvest thymocytes after opening the chest cavity of a naïve C57/Black-6 mouse, use curved fine-tip forceps to pull out the thymus. Place the organ into a tissue culture dish containing 10 milliliters of RPMI-1640 medium. Grind the whole thymus against the frosted ends of 2 microscope slides, and filter the resulting tissue suspension through a 70-micrometer cell strainer into a 50-milliliter tube. Collect the thymocytes by centrifugation, and resuspend the pellet in 40 milliliters.

After counting, centrifuge the cells again, and resuspend up to 2 x 10⁸ cells in 20 milliliters of fresh PBS per 50-milliliter tube. Label the cells with 5-micromolar CFSE in 20 milliliters of PBS per tube. Invert the tubes two to three times to mix, before incubating the thymocytes for no more than 2 minutes at room temperature, protected from light. At the end of the incubation, stop the reaction with 10 milliliters of heat-inactivated horse serum, and collect the cells by centrifugation.

Resuspend the labeled cells in 40 milliliters of fresh PBS for counting and centrifugation, followed by an additional wash with 40 milliliters of medium. After the medium wash, resuspend the thymocytes at a 7 x 10⁶ cells per milliliter of tissue culture medium concentration, and seed the cells in a 100-millimeter tissue culture dish. Then, add staurosporine to the cell culture at a 1-micromolar final concentration, and place the plate in a tissue culture incubator for 4 hours.

For peritoneal macrophage isolation, inject two C57/Black-6 mice intraperitoneally with 1 milliliter of 3% aged thioglycolate at day 0, before opening the abdominal skin without disturbing the peritoneum on day 5.

To flush the peritoneal cavity, use a 10-milliliter syringe equipped with an 18-gauge needle to quickly push 10 milliliters of wash buffer into the cavity before slowly aspirating the wash buffer for collection into a 50-milliliter tube. Wash the peritoneal gavage two times with PBS. Resuspend the peritoneal macrophages at a 2 times 10 to the sixth cells per milliliter of tissue culture medium concentration.

Next, seed 500 microliters of macrophages into each well of a 24-well plate, and place the plate in a tissue culture incubator for 2 hours. At the end of the incubation, aspirate the supernatant to remove the floating cells, and add 500 microliters of tissue culture medium to each well.

Immediately add 0 to 12 x 10⁶ thymocytes in 500 microliters of medium to each well of the culture, and place the plate in the tissue culture incubator for 4 hours. At the end of the incubation, wash each well two times with fresh PBS to remove any free-floating apoptotic cells, followed by a single wash with staining buffer. Next, label the plate-bound macrophages with 200 microliters of staining buffer supplemented with an appropriate anti-CD11b antibody per well, and place the plate at 4 degrees Celsius for 20 minutes.