eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of CFSE-labeled apoptotic thymocytes
<ol>
	<li>Euthanize two na&#239;ve C57/B6 mice by CO2 inhalation for 10 min and dissect to open the chest cavity, remove (pull out) the thymus with curved fine-tip forceps into tissue culture petri dish containing 10 mL of RPMI1640 medium.</li>
	<li>Obta...

analysis of efferocytosis of apoptotic thymocytes by peritoneal macrophages

Source: Zhen, Y. et al., <a href="https://app.jove.com/v/59731">Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages.</a> J. Vis. Exp. (2019)
This video demonstrates a procedure for analyzing peritoneal macrophage-mediated efferocytosis of apoptotic mouse thymocytes by fluorescence microscopy.

<img alt="Figure 1" src="/files/ftp_upload/21786/21786_Figure1.jpg" /> 
Figure 1. Percentage of phagocytosis of apoptotic thymocytes by peritoneal macrophages. Apoptotic thymocytes were induced by incubating with 1 &#956;M of staurosporine for 4 h and added into macrophage culture at a ratio as indicated in the figure panels: (A) 1:0; (B) 1:1; (C) 1:2; (D) 1:4; (E) 1:6; (F) 1:8; (G) 1:10; (H) 1:12. Data were acquired with a flow cytometer. The percen...

Analysis of Efferocytosis of Apoptotic Thymocytes by Peritoneal Macrophages

Source: Zhen, Y. et al., Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages. J. Vis. Exp. (2019)
This video demonstrates a procedure ...

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&#39;s outer leaflet, functioning as &#34;eat-me signals.&#34;
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.

Analysis of Efferocytosis of Apoptotic Thymocytes by Peritoneal Macrophages

Overview