eoe sub articles

EoE Sub Articles

1. Mtb infection of monocyte-derived cells
NOTE: The following steps must be performed in a BSL-3 facility.
<ol>
	<li>Resuspend the Mycobacterium tuberculosis (Mtb) pellet in serum-free RPMI medium in a new sterile 50 mL tube and adjust the final bacterial concentration to approximately 5 x 106 CFU/mL.</li>
	<li>Remove the cell culture medium from the 6-well plate(s) containing monocyte-derived cells. Add 1 mL of serum-free RPMI medium to each well. Add 1 mL of bacterial suspension per well to obtain a multiplicity of infection (MOI) 5:1, i.e., 5 x 106 CFU per 106 macrophages in 2 mL/well and incubate the plates for 4 h at 37&#176;C and 5% CO2.</li>
	<li>After infection, wash the cells 3 times with 1 mL of sterile wash buffer to remove extracellular bacteria. Tilt the plate and carefully remove the entire wash buffer from the corners. Resuspend the Mtb-infected monocyte-derived cells in 2 mL of RPMI complete medium without antibiotics and proceed to flow cytometry staining or incubate the cells for another 24 h (or other time-points) before flow cytometry.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>8-well chamber slides</td>
			<td>Lab-Tek</td>
			<td>154534</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Life Technologies Corporation</td>
			<td>SH30096.01</td>
			<td></td>
		</tr>
		<tr>
			<td>IFN-&#947;</td>
			<td>Peprotech</td>
			<td>300-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Na-pyruvate</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH300239.01</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30034.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 6-well Flat Bottom plates</td>
			<td>Corning Life Sciences</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>GM-CSF</td>
			<td>Peprotech</td>
			<td>300-03</td>
			<td></td>
		</tr>
	</tbody>
</table>

a fully differentiated macrophage infection model of monocyte-derived cells with mycobacterium tuberculosis

Source: Mily, A., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61807">Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection.</a> J. Vis. Exp.(2020).
This video demonstrates a method to study the responses to Mycobacterium tuberculosis infection in human M1 and M2 polarized monocyte-derived macrophages. The use of flow cytometry enables the study of a variety of surface markers associated with infected and uninfected macrophage populations.

A Fully Differentiated Macrophage Infection Model of Monocyte-Derived Cells with Mycobacterium tuberculosis

Source: Mily, A., et al. Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis ...

Obtain a culture of fully differentiated macrophages in a multi-well plate. These macrophages have surface markers, such as toll-like receptors or TLR-2 &#8212; a pattern recognition receptor that identifies various microbial components.
Add an appropriate amount of fluorescently labeled Mycobacterium tuberculosis or Mtb cell suspension into each well and incubate.
TLR-2 recognizes lipoarabinomannan, or LAM, a complex glycolipid component of the Mtb cell wall, initiating phagocytosis. The macrophage internalizes Mtb, sequestering the bacterium within a phagosome &#8212; a membrane-bound compartment.
The phagosome matures, by fusing with lysosomes, forming a phagolysosome &#8212; an acidic and enzymatic-rich compartment.
Within the phagolysosome, the Mtb inhibits the vacuolar-type proton pump responsible for generating an acidic pH. Consequently, the pH within the phagolysosome becomes near-neutral, deactivating enzymes that degrade engulfed pathogens.
The interaction between LAM and TLR2, also affects host cell signaling pathways, leading to the modulation of surface markers involved in antigen presentation.
The inactivation of phagolysosome and inhibition of host immune response enable Mtb to persist and replicate within the macrophage.

a-fully-differentiated-macrophage-infection-model-monocyte-derived

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

In Vitro Models

139 visualizações. Fonte: Mily, A., et al. Polarização de células derivadas de monócitos humanos M1 e M2 e análise com citometria de fluxo na infecção por Mycobacterium tuberculosis. J. Vis.(2020). Este vídeo demonstra um método para estudar as respostas à infecção por Mycobacterium tuberculosis em macrófagos humanos derivados de monócitos polarizados M1 e M2. O uso da citometria de fluxo permite o estudo de uma variedade de marcadores de superfície associados a populações de macrófagos infectad...

Vídeo: Um modelo de infecção de macrófagos totalmente diferenciado de células derivadas de monócitos com Mycobacterium tuberculosis - Conceito

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

Caracterização metabólica de Polarizada M1 e M2 macrófagos da medula óssea derivadas usando análises em tempo real do fluxo extracelular

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

JoVE Journal

Imunologia e Infecção

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Caracterização de derivados de monócitos células dendríticas humanas por imagem Citometria de Fluxo: Uma Comparação entre dois protocolos de isolamento de monócitos

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Isolation, Transfection, and Culture of Primary Human Monocytes

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4+ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14+ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.

Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.

Isolamento, Transfecção e Cultura dos Monócitos Humanos Primários

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. ...

A Fully Differentiated Macrophage Infection Model of Monocyte-Derived Cells with Mycobacterium tuberculosis

Transcrição

A Fully Differentiated Macrophage Infection Model of Monocyte-Derived Cells with Mycobacterium tuberculosis