Name: An Assay to Study the Regulation of Macrophage Phagocytosis by Mesenchymal Stem Cells
Uploaded: 2023-10-31T11:45:31
Description: NOTE: All medium preparation and cell culture techniques are carried out under aseptic conditions using a biosafety cabinet with laminar flow. All culture ...

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NOTE: All medium preparation and cell culture techniques are carried out under aseptic conditions using a biosafety cabinet with laminar flow. All culture incubation steps described are carried out using an incubator designed to maintain an atmosphere of 37 &#176;C, 5% CO2, and 95% humidity.
1. Cell culture
<ol>
	<li>Preparation of growth medium
	<ol>
		<li>For MSC (mesenchymal stem cells) and LADMAC (mouse bone marrow-derived macrophages; it is d...

an assay to study the regulation of macrophage phagocytosis by mesenchymal stem cells

Source: Evans, J. F., et al. <a href="https://app.jove.com/v/62729">Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging.</a> J. Vis. Exp. (2021).
This video demonstrates an assay to study mesenchymal stem cell (MSC) regulation of macrophage phagocytosis in a co-culture. Upon co-culturing MSC with macrophages, non-opsonized zymosan particles conjugated to a pH-sensitive fluorophore are added to be phagocytosed by the macrophages. With the increase in phagocytosis, the fluorescence intensity rises, allowing the determination of MSC-mediated regulation of phagocytic activity.

<img alt="Figure 1" src="/files/ftp_upload/21666/21666_Figure1.jpg" /> 
Figure 1: Overview of the co-culture and phagocytosis assay workflow. An outline of the workflow for quantitative and qualitative analysis of M&#934; phagocytosis activity in co-culture with MSC.
Supplementary File 1: A representative spreadsheet file of raw kinetic data from an optimal experiment. <a href="https://www.jove.com/files/ftp_upload/21666/2...

An Assay to Study the Regulation of Macrophage Phagocytosis by Mesenchymal Stem Cells

NOTE: All medium preparation and cell culture techniques are carried out under aseptic conditions using a biosafety cabinet with laminar flow. All culture ...

Take a multiwell plate with mesenchymal stem cells, or MSCs, in selected wells.
Introduce macrophages and incubate, facilitating MSC-macrophage interaction in the co-culture wells.
Add zymosan &#8212; a yeast cell wall component &#8212; conjugated to a pH-sensitive fluorophore to all the wells.
Macrophage scavenger receptors bind to zymosan, triggering its engulfment inside a phagosome, which fuses with a lysosome to form a phagolysosome.
An acidic pH inside the phagolysosome causes the fluorophore to emit fluorescence.
In the co-culture wells, MSCs project tunneling nanotubes, or TNT, cytoplasmic extensions that bridge with macrophages. MSC mitochondria travel through the TNT to enter the macrophages, increasing the total number of functional mitochondria.
An elevated mitochondrial number augments the production of adenosine triphosphate or ATP, which fuels the energy requirement for an increase in phagocytosis.
Record the fluorescence intensity over time from the internalized fluorophore-conjugated zymosans.
Co-culture shows increased fluorescence compared to wells with only macrophages, indicating MSC-mediated enhancement of macrophage phagocytosis.

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Research

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host Defense Mechanism

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major immune cells and only phagocytes in the brain, recent studies have shown that astrocytes also participate in various phagocytic processes, such as developmental synapse elimination and clearance of amyloid beta plaques in Alzheimer&#39;s disease (AD). Despite these findings, the efficiency of astrocyte engulfment and degradation of their targets is unclear compared with that of microglia. This lack of information is mostly due to the lack of an assay system in which the kinetics of astrocyte- and microglia-mediated phagocytosis are easily comparable. To achieve this goal, we have developed a long-term live-imaging in vitro phagocytosis assay to evaluate the phagocytic capacity of purified astrocytes and microglia. In this assay, real-time detection of engulfment and degradation is possible using pH indicator-conjugated synaptosomes, which emit bright red fluorescence in acidic organelles, such as lysosomes. Our novel assay provides simple and effective detection of phagocytosis through live-imaging. In addition, this in vitro phagocytosis assay can be used as a screening platform to identify chemicals and compounds that can enhance or inhibit the phagocytic capacity of astrocytes. As synaptic pruning malfunction and pathogenic protein accumulation have been shown to cause mental disorders or neurodegenerative diseases, chemicals and compounds that modulate the phagocytic capacity of glial cells should be helpful in treating various neurological disorders.

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

This protocol presents an in vitro live-imaging phagocytosis assay to measure the phagocytic capacity of astrocytes. Purified rat astrocytes and microglia are used along with pH indicator-conjugated synaptosomes. This method can detect real-time engulfment and degradation kinetics and provides a suitable screening platform to identify factors modulating astrocyte phagocytosis.

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

JoVE Journal

Neuroscience

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

Immunology and Infection

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

An Assay to Study the Regulation of Macrophage Phagocytosis by Mesenchymal Stem Cells

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