Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Our body generates one to 10 billion of apoptotic cells every day. The efficient clearance of these cells helps in removing cell debris and maintaining inventories. This method may be used in many applications to characterize apoptotic cell binding and ingestion by many other phagocytic cell types.

Demonstrating this procedure will be Yuxuan Zhen, a senior technician in my lab. To harvest thymocytes, after opening the chest cavity of a naive 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 two 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 two times 10 to the eight cells in 20 milliliters of fresh PBS per 50 milliliter tube.

Label the cells with five 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 two 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 seven times 10 to the six 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 one micromolar final concentration and place the plate in a tissue culture incubator for four hours.

For peritoneal macrophage isolation, inject two C57 black 6 mice intraperitoneally with one milliliter of 3%aged thioglycollate at day zero before opening the abdominal skin without disturbing the peritoneum on day five. 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 lavage two times with PBS.

Resuspend the peritoneal macrophages at a two times 10 to the six 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 two 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 zero to 12 times 10 to the six thymocytes in 500 microliters of medium to each well of the culture and place the plate in the tissue culture incubator for four 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 four degrees Celsius for 20 minutes.

In this representative experiment, up to 30%of the thioglycollate stimulated peritoneal macrophages demonstrated a positive signal in the CFSE channel indicating that these phagocytes had ingested CFSE-labeled apoptotic cells. Microscopic observation of macrophage engulfment of apoptotic cells is essential for investigating the capacity of macrophages to ingest apoptotic cells as CFSE positive macrophages spread out in the bottom right quadrant due to different intensities indicating that the number of apoptotic cells within individual macrophages is different. A higher ratio of apoptotic cells to macrophages not only increases the number of macrophages ingesting apoptotic cells but also enhances the ability of the macrophages to ingest more apoptotic cells.

In another set of experiments, about 15%of the macrophages became CFSE positive when CFSE labeled apoptotic thymocytes were added to the macrophage culture for four hours indicating that these macrophages were the phagocytic macrophages. Mer antibody blocks macrophage efferocytosis in a dose-dependent manner and the overall blockage may account for about 30%of the efferocytosis efficiency in the current setting. In addition, a newly approved TAM receptor inhibitor attenuates macrophage efferocytosis in a dose-dependent manner up to an about 100 nanomolar concentration.

Always remember to check the percentage of apoptotic cells as this number directly affects the efficiency of efferocytosis. The phagocytic macrophages can be further analyzed by Western blot to investigate the signaling molecules regulated by engulfment process and by microscopy to study the dynamics of engulfment.