eoe sub articles

EoE Sub Articles

1. Antigenic preparation
NOTE: Three different antigenic preparations can be used to assess the immune response to Hi. These are 1) a subcellular component (typically from the bacterial cell wall); 2) killed and inactivated bacteria; and 3) live bacteria. Determine the use of each antigenic preparation prior to the initiation of any experiments.
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	<li>Subcellular components
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		<li>Obtain subcellular components from sources, including commercial prep...

an in vitro technique to stimulate lymphocytes via pathogenic bacteria

Source: Dousha, L., et al. <a href="https://app.jove.com/v/62572">Assessing Respiratory Immune Responses to Haemophilus Influenzae.</a> J. Vis. Exp. (2021)
This video demonstrates an in vitro technique to stimulate lymphocytes via non-typeable Haemophilus influenzae (NTHi) and to assess the immune response. Incubating isolated peripheral blood mononuclear cells (PBMCs) with NTHi causes the bacterial cells to stimulate T lymphocytes, leading to cytokine production. Upon inhibiting the secretion of cytokines, immunofluorescence staining is performed to detect the accumulated intracellular cytokines in stimulated T lymphocytes.

<img alt="Figure 1" src="/files/ftp_upload/21620/21620_Figure1.jpg" /> 
Figure 1: Cytokine production in lung tissue. Cells are first analyzed for their expression of the leukocyte marker CD45 (A) using flow cytometry. This population is then analyzed further for CD3 expression (B) and CD4/CD8 expression (C). CD3/CD4+ cells are assessed for intracellular cytokine production in control (D) and NTHi-st...

An In Vitro Technique to Stimulate Lymphocytes via Pathogenic Bacteria

Source: Dousha, L., et al. Assessing Respiratory Immune Responses to Haemophilus Influenzae. J. Vis. Exp. (2021)
This video demonstrates an in vitro ...

To stimulate lymphocytes via Haemophilus influenzae &#8212; a pathogenic bacterium, take a suspension of isolated peripheral blood mononuclear cells, or PBMCs, including lymphocytes and antigen-presenting cells or APCs.
Add a bacterial suspension and incubate at physiological temperature.
Pattern recognition receptors on the APCs bind to specific components of the bacterial cells, triggering phagocytosis. The phagosome &#8212; containing internalized bacteria &#8212; fuses with the lysosome, causing bacterial degradation and processing into peptide fragments.
Major histocompatibility complex or MHC molecules bind to the peptide fragments and present them on the cell surface as antigens. T lymphocytes bind to the presented antigen via the T cell receptor.
Add co-stimulatory antibodies that bind to specific surface receptors on T lymphocytes. The synergistic binding induces downstream signaling, activating T lymphocyte cytokine production.
Add a Golgi-blocking compound &#8212; blocking vesicular protein transport from the endoplasmic reticulum to the Golgi &#8212; to inhibit cytokine secretion from T lymphocytes, facilitating intracellular cytokine accumulation.
Add a fixative to preserve cellular morphology and detergent to permeabilize the cell membrane. Add a cocktail of fluorescently labeled antibodies, including antibodies binding to the T lymphocyte-specific surface markers &#8212; labeling the cell surface &#8212; and cytokine-specific antibodies that penetrate inside the cell to bind intracellular cytokines.
Perform flow cytometry to detect cells with dual fluorescence &#8212; indicating cytokine production by stimulated T lymphocytes.

an-in-vitro-technique-to-stimulate-lymphocytes-via-pathogenic-bacteria

Research

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Immune Modulation

This video demonstrates an in vitro technique to stimulate lymphocytes via non-typeable Haemophilus influenzae (NTHi) and to assess the immune response. Incubating isolated peripheral blood mononuclear cells (PBMCs) with NTHi causes the bacterial cells to stimulate T lymphocytes, leading to cytokine production. Upon inhibiting the secretion of cytokines, immunofluorescence staining is performed to detect the accumulated intracellular cytokines in stimulated T lymphocytes.

Overview

Protocol

<img title="figure-representative results-58" alt="figure-representative results-58" src="/files/ftp_upload/21620/21620_Figure1.jpg" /> 
Figure 1: Cytokine production in lung tissue. Cells are first analyzed for their expression of the leukocyte marker CD45 (A) using flow cytometry. This population is then analyzed further for CD3 expression (B) and CD4/CD8 expression (C). CD3/CD4+ cells are assessed for intracellular cytokine production in control (D) and NTHi-st...

An In Vitro Technique to Stimulate Lymphocytes via Pathogenic Bacteria

In This Article

Overview

Protocol

Representative Results