eoe sub articles

EoE Sub Articles

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Isolation of human monocytes and differentiation into macrophages
<ol>
	<li>Obtain anticoagulated blood from de-identified healthy individuals at a regional blood bank and isolate peripheral blood mononuclear cells (PBMCs) as indicated below. 
	<st...

visualization of caspase activation in human monocyte-derived macrophages

Source: Bol&#237;var, B. E., et. al., <a href="https://app.jove.com/v/63162">Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages.</a> J. Vis. Exp. (2022).
This video demonstrates the activation of caspase proteins in human macrophages using bimolecular fluorescence complementation reporter proteins. The pro-domains of caspase-1 are fused with non-fluorescent fragments of Venus proteins, which are recruited to inflammasome complexes. The proximity of inflammasomes induces the refolding and fluorescence of Venus fragments, confirming caspase activation in the macrophages.

<img alt="Figure 1" src="/files/ftp_upload/21616/21616_Figure1.jpg" /> 
Figure 1: Schematic overview of the experimental workflow.
<img alt="Figure 1" src="/files/ftp_upload/21616/21616_Figure2.jpg" /> 
Figure 2: Differentiation of CD14-monocytes and transfection of human MDM. (A) Representative bright-field images of CD14+ monocytes from peripheral...

Visualization of Caspase Activation in Human Monocyte-Derived Macrophages

Source: Bol&#237;var, B. E., et. al., Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages. J. Vis. Exp. 
...

In immune cells, caspase-1, an inflammatory caspase, is a key immune-responsive component that exists in an inactive form. These inactive forms consist of a pro-domain fused with a catalytic domain and become active via proximity-induced dimerization.
To visualize caspase activation in vivo, begin with a culture of transfected human monocyte-derived macrophages expressing red fluorescent proteins and a pair of fluorescent complementation reporter proteins. The reporter proteins contain a pro-domain of caspase-1 fused with non-fluorescent fragments of the Venus protein, either Venus-N or Venus-C.
Treat the cells with an imaging medium containing lipopolysaccharides &#8212; a bacterial-derived inflammatory stimulant, and potassium ionophores &#8212; nigericin.
Lipopolysaccharides interact with pattern recognition receptors on the macrophage, initiating the assembly of an inflammasome complex.
Nigericin molecules enter the cell and bind to potassium ions, causing their efflux into the extracellular medium and decreasing the intracellular potassium ion concentration. This leads to the priming of inflammasome sensor proteins with adaptor proteins &#8212; a bridging protein between sensor protein and procaspase-1, forming the complex.
The pro-domains containing Venus fragments interact with the complex&#39;s adaptor proteins. This brings the two fragments in proximity, forcing them to refold and fluoresce.
Visualize the cells under an epifluorescence microscope.
The transfected cells appear red with green fluorescence spots, confirming caspase activation.

visualization-caspase-activation-human-monocyte-derived

Research

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Immune Modulation

This video demonstrates the activation of caspase proteins in human macrophages using bimolecular fluorescence complementation reporter proteins. The pro-domains of caspase-1 are fused with non-fluorescent fragments of Venus proteins, which are recruited to inflammasome complexes. The proximity of inflammasomes induces the refolding and fluorescence of Venus fragments, confirming caspase activation in the macrophages.

Overview

Protocol

<img title="figure-representative results-68" alt="figure-representative results-68" src="/files/ftp_upload/21616/21616_Figure1.jpg" /> 
Figure 1: Schematic overview of the experimental workflow.
<img title="figure-representative results-292" alt="figure-representative results-292" src="/files/ftp_upload/21616/21616_Figure2.jpg" /> 
Figure 2: Differentiation of CD14-monocytes and transfection of human MDM. (A) Representative bright-field images of CD14+ monocytes from peripheral...

Visualization of Caspase Activation in Human Monocyte-Derived Macrophages

In This Article

Overview

Protocol

Representative Results

Reprints and Permissions