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EoE Sub Articles

1. Preparing the solutions
<ol>
	<li>Prepare L929-conditioned media.
	<ol>
		<li>Plate 1 &#215; 106&#160;L929 cells (see&#160;Table of Materials) in an 182 cm2&#160;tissue culture flask containing 50 mL of L929 culture media (see&#160;Table 1&#160;for the preparation of the media).</li>
		<li>Grow the cells in a humidified incubator at 37 &#176;C with 5% carbon dioxide, CO2.</li>
		<li>After 7 days, collect the supernatant, and filter u...

assessment of the activation of multiple caspases in macrophages

Source: Han, J. et al., <a href="https://app.jove.com/v/64308">Evaluation of Caspase Activation to Assess Innate Immune Cell Death.</a>&#160;J. Vis. Exp.&#160;(2023)
This video demonstrates the assessment of multiple caspase activations in bone marrow-derived macrophages through stimulation with the influenza virus. Following macrophage lysis, heat-inactivation of lysate, and centrifugation, intracellular caspases are collected for subsequent immunological analysis.

Assessment of the Activation of Multiple Caspases in Macrophages

1. Preparing the solutions

	Prepare L929-conditioned media.
	
		Plate 1 &#215; 106&#160;L929 cells (see&#160;Table of Materials) in an 182 ...

Begin with a multi-well plate containing adhered bone marrow-derived macrophages.
Introduce influenza A viruses that fuse with macrophages, releasing ribonucleoproteins containing viral RNA.
The macrophages&#39; Z-DNA binding protein 1, or ZBP1 interacts with viral ribonucleoproteins, triggering ZBP1 oligomerization, with kinases and caspase 8.
The oligomerized ZBP1 further recruits inflammasome sensor proteins, adaptor proteins, and procaspase-1, forming a multimeric complex &#8212; a PANoptosome.
Within the PANoptosome, initiator caspase-8 activates through proximity cleavage, further activating effector caspases, including caspase-3 and 7.
Additionally, the inflammasome sensor proteins within the PANoptosome activate a signaling cascade, converting pro-caspase-1 to its active form, caspase-1.
Concurrently, viral infection induces the release of cytochrome-c from the mitochondria, triggering apoptosome activation, and converting pro-caspase-9 to its active form.
Viral infection also triggers the non-canonical inflammasome pathway, activating caspase-11.
Introduce a lysis buffer to lyse the macrophages; collect the lysate, and heat it to inactivate proteases, preventing caspases&#39; degradation.
Centrifuge and collect the supernatant containing multiple caspases for further analysis.

assessment-of-the-activation-of-multiple-caspases-in-macrophages

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first to be activated in these pathways. This group includes caspase-2, -8, and -9, as well as the inflammatory caspases, caspase-1, -4, and -5. The initiator caspases are all activated by dimerization following recruitment to specific multiprotein complexes called activation platforms. Caspase Bimolecular Fluorescence Complementation (BiFC) is an imaging-based approach where split fluorescent proteins fused to initiator caspases are used to visualize the recruitment of initiator caspases to their activation platforms and the resulting induced proximity. This fluorescence provides a readout of one of the earliest steps required for initiator caspase activation. Using a number of different microscopy-based approaches, this technique can provide quantitative data on the efficiency of caspase activation on a population level as well as the kinetics of caspase activation and the size and number of caspase activating complexes on a per cell basis.

This protocol describes caspase Bimolecular Fluorescence Complementation (BiFC); an imaging-based method that can be used to visualize induced proximity of initiator caspases, which is the first step in their activation.

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first ...

JoVE Journal

Biochemistry

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...

Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection

Caspases, a family of cysteine proteases, orchestrate programmed cell death in response to various stimuli, including microbial infections. Initially described to occur by apoptosis, programmed cell death is now known to encompass three interconnected pathways: pyroptosis, apoptosis, and necroptosis, together coined as one process, PANoptosis. Influence A virus (IAV) infection induces PANoptosis in mammalian cells by inducing the activation of different caspases, which, in turn, cleave various host as well as viral proteins, leading to processes like the activation of the host innate antiviral response or the degradation of antagonistic host proteins. In this regard, caspase 3-mediated cleavage of host cortactin, histone deacetylase 4 (HDAC4), and histone deacetylase 6 (HDAC6) has been discovered in both animal and human epithelial cells in response to the IAV infection. To demonstrate this, inhibitors, RNA interference, and site-directed mutagenesis were employed, and, subsequently, the cleavage or resistance to cleavage and the recovery of cortactin, HDAC4, and HDAC6 polypeptides were measured by western blotting. These methods, in conjunction with RT-qPCR, form a simple yet effective strategy to identify the host as well as viral proteins undergoing caspase-mediated cleavage during an infection of IAV or other human and animal viruses. The present protocol elaborates the representative results of this strategy, and the ways to make it more effective are also discussed.

Influenza A virus (IAV) infection activates the caspases that cleave host and viral proteins, which, in turn, have pro- and antiviral functions. By employing inhibitors, RNA interference, site-directed mutagenesis, and western blotting and RT-qPCR techniques, caspases in infected mammalian cells that cleave host cortactin and histone deacetylases were identified.

Caspases, a family of cysteine proteases, orchestrate programmed cell death in response to various stimuli, including microbial infections. Initially ...

Assessment of the Activation of Multiple Caspases in Macrophages

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