Analysis of Efferocytosis of Apoptotic Thymocytes by Peritoneal Macrophages
Transcript
Efferocytosis is the process by which phagocytic cells clear apoptotic cells.
To analyze efferocytosis, obtain apoptosis-susceptible mouse thymocytes. Incubate with cell-permeable CFSE dye.
Intracellular esterases cleave CFSE to cell-impermeable fluorescent dye, staining thymocytes. Add heat-inactivated serum to prevent excess staining.
Plate the stained thymocytes on a culture plate. Add staurosporine, a protein kinase inhibitor, which disrupts the critical cellular signaling cascade, inducing apoptosis. Further, phosphatidylserine residues translocate to the cell membrane's outer leaflet, functioning as "eat-me signals."
Add increasing apoptotic thymocyte concentrations to multi-well plate wells containing activated mouse peritoneal macrophages, which express specific receptor tyrosine kinases.
These kinases bind the exposed phosphatidylserine on apoptotic thymocytes, facilitating their internalization by macrophages.
Post-incubation, remove free-floating apoptotic thymocytes. Add fluorophore-tagged anti-CD11b antibodies, which bind to macrophage CD11b molecules.
Using a fluorescence microscope, observe fluorescent apoptotic thymocytes within differently fluorescent macrophages, indicating efferocytosis.
Wells with higher apoptotic thymocyte concentrations display increased internalized thymocytes.
