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1. LDH Release Assay
NOTE: For the LDH release assay, seed the cells in a 96-well plate. Do not remove the medium after priming.
<ol>
	<li>Replace the medium with fresh medium (DMEM-5: 10 mL of phenol-red free DMEM + 5 mM HEPES + 0.2 mg/mL L-glutamine + 0.05 mM 2-mercaptoethanol + 5% FBS) containing 100 ng/mL LPS in wells containing adherent macrophage cells (500 &#956;L for the 24-well plates or 50 &#956;L for the 96-well plates). 
	NOTE: There are a variety of structural variations in LPS, which affect the ability to stimulate TLR4-mediated priming. LPS from Salmonella minnesota R595 (Re) is recommended.</li>
	<li>Incubate the plate for 3 h at 37 &#176;C and 5% CO2.</li>
	<li>Add 50 &#956;L of DMEM-5 containing 10 &#956;M nigericin to the experimental wells and 50 &#956;L DMEM-5 to the spontaneous and 100% lysis control wells. Incubate for 60 min at 37 &#176;C and 5% CO2.</li>
	<li>After 30 min, add 10 &#956;L of 10x lysis buffer to the 100% lysis control wells and add 10 &#956;L of DMEM-5 to the other wells. During this incubation, remove 1 vial of substrate mix (1.5 mL of freeze-dried substrate) and 1 aliquot of assay buffer (~12 mL) from the freezer and let them thaw protected from light to room temperature. 
	NOTE: The substrate mix contains a tetrazolium salt (iodonitrotetrazolium violet), which is converted to a red formazan product.</li>
	<li>After the 60 min total incubation, centrifuge the plate for 5 min at 500 x g at 10 &#176;C. Transfer 50 &#956;L of the supernatant to a clear flat-bottom plate. During this time, add 12 mL of assay buffer to the vial of substrate mix and incubate at room temperature for 5 min before using.</li>
	<li>Add 50 &#956;L of substrate to each well and incubate for 30 min in the dark. Include medium-only wells for blank values. Check the plate after 15 min to make sure that the signal will not exceed the detection limit of the plate reader.</li>
	<li>After 30 min, add 50 &#956;L of stop solution (1 M acetic acid) and measure OD490 using a plate reader.</li>
	<li>Calculate the percentage of cell lysis using the following formula: 
	% Cytotoxicity = ((Experimental - Spontaneous)/(Maximum - Spontaneous))&#215;100 
	Experimental: nigericin-treated cells; spontaneous: untreated cells; maximum: cells exposed to lysis buffer.</li>
</ol>

a lactate dehydrogenase release assay to detect macrophage death following nigericin exposure

Source: den Hartigh, A.B. et al., <a href="https://app.jove.com/v/57463">Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages.</a> J. Vis. Exp. (2018)
This video demonstrates the lactate dehydrogenase assay to detect inflammasome-mediated macrophage death. The macrophages are exposed to lipopolysaccharide for priming, followed by exposure to nigericin to cause inflammasome-induced cell death, which is determined by the lactate dehydrogenase assay.

A Lactate Dehydrogenase Release Assay To Detect Macrophage Death Following Nigericin Exposure

1. LDH Release Assay
NOTE: For the LDH release assay, seed the cells in a 96-well plate. Do not remove the medium after priming.

	Replace the medium with ...

Take an assay plate containing macrophages primed with lipopolysaccharide.
Priming increases the expression of inactive pro-inflammatory cytokine precursors and NLRP3 proteins.
Add nigericin, a potassium ionophore, promoting ion efflux. This disrupts intracellular potassium levels, triggering NLRP3 proteins, to form a large complex.
Oligomerized NLRP3 recruits the adaptor proteins, ASC, causing their polymerization. ASC filaments recruit pro-caspase-1, forming NLRP3-inflammasome, a multiprotein complex.
Pro-caspase-1 undergoes proximity-induced auto-activation, generating caspase-1. Caspase-1&#160; cleaves the pro-inflammatory cytokine and gasdermin-D protein, creating a plasma membrane pore.
This results in cytokine secretion and inflammatory programmed cell death, pyroptosis, releasing intracellular contents, including lactate dehydrogenase, or LDH. Collect the LDH-containing supernatant.
Add substrate mixture &#8212; lactate, iodonitrotetrazolium violet, or INT dye, NAD+, and diaphorase. LDH oxidizes lactate to pyruvate while reducing NAD+. Diaphorase catalyzes the reduction of INT, forming red-colored formazan.
The color intensity corresponds to LDH release, with elevated levels indicating increased pyroptosis-associated cell damage and death.

a-lactate-dehydrogenase-release-assay-to-detect-macrophage-death

Research

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host Defense Mechanism

128 Views. Source: den Hartigh, A.B. et al., Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages. J. Vis. Exp. (2018) This video demonstrates the lactate dehydrogenase assay to detect inflammasome-mediated macrophage death. The macrophages are exposed to lipopolysaccharide for priming, followed by exposure to nigericin to cause inflammasome-induced cell death, which is determined by the lactate dehydrogenase assay. 

Video: A Lactate Dehydrogenase Release Assay To Detect Macrophage Death Following Nigericin Exposure - Concept

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development and implementation of biomedical devices and tissue engineering constructs. Macrophages, an innate immune cell, are key players in the foreign body reaction because they remain at the implant site for the lifetime of the device, and are commonly studied to gain an understanding of this detrimental host response. Many biomaterials researchers have shown that adsorbed protein layers on implanted materials influence macrophage behavior, and subsequently impact the host response. The methods in this paper describe an in vitro model using adsorbed protein layers containing cellular damage molecules on polymer biomaterial surfaces to assess macrophage responses. An NF-&#1082;B/AP-1 reporter macrophage cell line and the associated colorimetric alkaline phosphatase assay were used as a rapid method to indirectly examine NF-&#1082;B/AP-1 transcription factor activity in response to complex adsorbed protein layers containing blood proteins and damage-associated molecular patterns, as a model of the complex adsorbed protein layers formed on biomaterial surfaces in vivo.

This protocol provides researchers with a rapid, indirect method of measuring TLR-dependent NF-&#1082;B/AP-1 transcription factor activity in a murine macrophage cell line in response to a variety of polymeric surfaces and adsorbed protein layers that model the biomaterial implant microenvironment.

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development ...

JoVE Journal

Bioengineering

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1&#946; and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1&#946; release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...

Immunology and Infection

Evaluation of Caspase Activation to Assess Innate Immune Cell Death

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is the initiation of innate immune programmed cell death (PCD) to eliminate infected or damaged cells and propagate immune responses. However, excess PCD is associated with inflammation and pathology. Therefore, understanding the activation and regulation of PCD is a central aspect of characterizing innate immune responses and identifying new therapeutic targets across the disease spectrum.
This protocol provides methods for characterizing innate immune PCD activation by monitoring caspases, a family of cysteine-dependent proteases that are often associated with diverse PCD pathways, including&#160;apoptosis,&#160;pyroptosis, necroptosis, and PANoptosis. Initial reports characterized caspase-2, caspase-8, caspase-9, and caspase-10 as initiator caspases and caspase-3, caspase-6, and caspase-7 as effector caspases in apoptosis, while later studies found the inflammatory caspases, caspase-1, caspase-4, caspase-5, and caspase-11, drive pyroptosis. It is now known that there is extensive crosstalk between the caspases and other innate immune and&#160;cell death molecules across the previously defined PCD pathways, identifying a key knowledge gap in the mechanistic understanding of innate immunity and PCD and&#160;leading to the characterization of PANoptosis. PANoptosis is a unique innate immune inflammatory PCD pathway regulated by PANoptosome complexes, which integrate components, including caspases,&#160;from other cell death pathways.
Here, methods for assessing the activation of caspases in response to various stimuli are provided. These methods allow for the characterization of PCD pathways both in vitro and in vivo, as activated caspases undergo proteolytic cleavage that can be visualized by western blotting using optimal antibodies and blotting conditions. A protocol and western blotting workflow have been established that allow for the assessment of the activation of multiple caspases from the same cellular population, providing a comprehensive characterization of the PCD processes. This method can be applied across research areas in development, homeostasis, infection, inflammation, and cancer to evaluate PCD pathways throughout cellular processes in health and disease.

This protocol describes a comprehensive method for assessing caspase activation (caspase-1, caspase-3, caspase-7, caspase-8, caspase-9, and caspase-11) in response to both in vitro and in vivo (in mice) models of infection, sterile insults, and cancer to determine the initiation of cell death pathways, such as pyroptosis, apoptosis, necroptosis, and PANoptosis.

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is ...

A Lactate Dehydrogenase Release Assay To Detect Macrophage Death Following Nigericin Exposure

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