eoe sub articles

EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Parasite culture
NOTE: Follow the local regulations for human pathogens handling.
<ol>
	<li>Maintain&#160;Plasmodium falciparum&#160;parasites according to the standard protocol as described before&#160;in the parasite culture medium (see&#160;Table of Materials).</li>
	<li>Keep the par...

an assay for quantifying antibody-mediated phagocytosis of parasite-infected erythrocytes

Source: Quintana, M. d. P. et al., <a href="https://app.jove.com/v/61538">Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay.</a>&#160;J. Vis. Exp.&#160;(2020)
This video demonstrates a flow cytometry-based phagocytosis assay involving labeled and opsonized Plasmodium falciparum-infected erythrocytes by monocytes. Flow cytometry identifies fluorescence in monocytes, confirming the successful identification of opsonized erythrocytes through phagocytosis.

<img alt="Figure 1" src="/files/ftp_upload/21901/21901_Figure1.jpg" /> 
Figure 1: Fc receptors expressed on the THP-1 cell surface. (A) Fc&#947;-receptor III/CD16 (red). (B) Fc&#947;-receptor II/CD32 (green).&#160;C.&#160;Fc&#947;-receptor I/CD64 (orange). Unlabeled cells are shown in blue.
<img alt="Figure 2" src="/files/ftp_upload/21901/21901_Figure2.jpg" /> 
Figure 2: Phagocytosis assay flow...

An Assay for Quantifying Antibody-Mediated Phagocytosis of Parasite-Infected Erythrocytes

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Parasite culture
NOTE: Follow the ...

Take Plasmodium falciparum-infected erythrocytes. Label the parasite DNA with a fluorescent dye for parasite visualization.
Incubate these parasite-infected erythrocytes with sera from malaria-exposed individuals. These cells display parasite antigens on their surface, which attach to naturally acquired human IgG antibodies in sera, opsonizing cells for phagocytosis.
Transfer the opsonized parasite-infected erythrocytes to a protein-coated multi-well plate containing a suspension of human-derived monocytes &#8212; or phagocytic cells.
During incubation, the Fc-gamma receptors on monocytes interact with the IgG-opsonized erythrocytes, triggering the engulfment of erythrocytes into phagosomes.
Post-incubation, centrifuge the plate to prevent further cellular interactions and stop phagocytosis.
Discard the supernatant. Treat with ammonium chloride lysing solution, selectively rupturing non-phagocytosed infected erythrocytes without affecting monocytes.
Add serum-containing buffer to stop the lysing process. Centrifuge and discard the supernatant containing lysed erythrocytes.
Resuspend the monocytes in a buffer.
Perform flow cytometry to quantify monocytes that display fluorescence from the parasite-infected erythrocytes within them, confirming their antibody-mediated phagocytosis.

an-assay-for-quantifying-antibody-mediated-phagocytosis-parasite

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

JoVE Journal

Immunology and Infection

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

An Assay for Quantifying Antibody-Mediated Phagocytosis of Parasite-Infected Erythrocytes

成績單