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Analysis of Efferocytosis of Apoptotic Thymocytes by Peritoneal Macrophages

Analysis of Efferocytosis of Apoptotic Thymocytes by Peritoneal Macrophages

Transcription

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.

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