Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Summary

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

Abstract

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 °C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Introduction

Macrophage phagocytosis assays are often used to study the innate immune function. The innate immune response may indicate susceptibility to infection. Macrophage cell lines are widely used in immunology studies. However, the extended passage may cause gene loss and compromised immune functions in these cell lines. Thus, the primary peritoneal macrophages are the ideal object in which to study the cell function¹.

Although the innate immune response was thought to be intact in the aged body, the phagocytic ability may decrease compared to that in the younger body^2,3....

Protocol

​All procedures were performed under the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, and the protocols were approved by the Animal Care and Use Committee of Dalian Medical University. Sixteen-month-old (with a body weight of 30-35 g) and eight-week-old (20-25 g) SPF (specific-pathogen-free) male C57BL/6 mice were obtained from the SPF animal center of Dalian Medical University. All mice were kept in animal housing with access to food and water ad libitum. The temperature was kept at 20-24 °C, humidity was 40%-70%, and lighting was 12 h light/12 h dark. Animals were allowed to acclimate to the environment for....

Results

The pET-SUMO vector utilizes a small ubiquitin-like modifier to allow the expression of native proteins in the E. coli. SUMO fusion can significantly enhance the EGFP solubility, allowing it to be detected easily. If the EGFP expression is successfully induced by lactose, green colonies can be observed in the dark (Figure 1A). Green dots, which represent the EGFP-expressing E. coli, can be observed under a fluorescence microscope using a 40x.......

Discussion

The steps in this protocol are quite simple and straightforward. One of the critical steps is to induce EGFP expression on E. coli. Usually, when a gene from eukaryotes, like EGFP, is planned to express in prokaryotes like E. coli, there is a risk that the protein will form inactive aggregates (inclusion bodies), which changes the protein's native structure and activity. By using the pET-SUMO vector and constructing the pET-SUMO-EGFP plasmid, the EGFP-SUMO fusion protein expressed successfully, and the .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BD FACSCanto II Flow cytometer	BD Biosciences	-
Biotin anti-mouse CD16/32 Antibody	Biolegend	Cat101303
Champion pET SUMO Protein Expression system	Invitrogen	K300-01
Custom Gene Synthesis Service	Takara Biotech.	-
DAPI(4',6-Diamidino-2-Phenylindole, Dihydrochloride)	ThermoFisher	D1306
F4/80-PE anti-mouse antibody for FACS	Biolegend	Cat123110
Leica DMI3000 B  Inverted Microscope	Leica Microsystems	-
PE Rat IgG2a, κ-isotype control	Biolegend	Cat400507
Phalloidin 633 fluorescence dye conjugated working solution	AAT Bioquest	Cat23125
Thioglycollate medium	Sigma-Aldrich	T9032