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Summary

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

Abstract

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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

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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. Here, we will demonstrate a method to assess the phagocytosis of peritoneal macrophages from young (eight-week-old) and aged (16-month-old) mouse using EGFP-expressing E. coli, which is convenient, quick, and economically feasible.

The use of an EGFP-expressing E. coli strain is one of the advantages of this assay because these bacteria are stable and display a strong fluorescence signal, even after macrophages are fixed by 4% (w/v) paraformaldehyde. Additionally, by using the EGFP-expressing E. coli, researchers do not need further staining after phagocytosis, which saves time. Furthermore, macrophages are immunoresponsive for E. coli surface antigen, making E. coli more suitable for the phagocytosis assay than using the EGFP-expressing fungi or fluorescein-labeled beads.

With EGFP-expressing E. coli, a phagocytosis assay can be easily accomplished in 2 h and measured by both flow cytometry and fluorescence microscopy, depending on the researcher’s purpose. Since this method directly measures the phagocytic ability, the results are more reproducible than other indirect methods.

This method has also been validated in a RAW264.7 cell line and human peripheral blood mononuclear cells⁴. The text below provides the detailed step-by-step instructions for performing this assay and highlights the critical steps that the researchers may modify to meet the needs of their experiments.

Protocol

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​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 at least 7 days before the experiment.

1. Construction of the pET-SUMO-EGFP plasmid and induction of the EGFP expression

1. Synthesize the EGFP gene fragment (a 717 bp sequence synthesized by a custom gene synthesis service, see Table of Materials and Supplementary File 1 for the sequence) and amplify the fragment with the forward primer (5'-ATGGTGAGCAAGGGCGAGGAGC-3') and reverse primer (5'-CTTGTACAGCTCGTCCATGCCG-3'), using high-fidelity Taq DNA polymerase.
2. To ensure that the polymerase chain reaction (PCR) products have single 3' adenine overhangs for the TA cloning in the next step, use a 30 min extension at 72 °C after the last cycle (see Supplementary File 1 for PCR conditions). Check the PCR product by agarose gel electrophoresis.
3. Clone the PCR product into the pET-SUMO vector (see Table of Materials) using the TA cloning method⁵ with T4 DNA ligase. Incubate the reaction at room temperature (20-25 °C) for 30 min. The vector is linearized between nucleotides 653 and 654 with a 1 bp 5′ T-overhang on each strand.
4. Transform the ligation product into the chemically competent BL21(DE) E. coli strain as follows: add 5 µL (100 ng) of PCR product to 100 µL of BL21(DE) competent cells via heat shock at 42 °C for 90 s; keep the mixture on ice for 3 min, and then, add 400 µL of lysogeny broth (LB) medium preheated at 37 °C, shaking it for 1 h at 37 °C and 120 rpm.
5. Inoculate 100 µL of the bacteria onto the surface of an LB-kanamycin (100 µg/mL) plate with the inducer lactose (0.5 mmol/L), yielding the EGFP expression strain. Incubate the plate at 37 °C overnight.
   NOTE: If the EGFP is successfully expressed, some colonies can be observed as glowing green light in the dark.
   1. Optionally, select colonies to verify the inserted EGFP fragment by DNA sequencing. The primers for DNA sequencing are: forward, 5'-AGATTCTTGTACGACGGTATTAG-3'; reverse, 5'-TAGTTATTGCTCAGCGGTGG-3'.
6. Inoculate a positive colony into 5 mL of LB medium with 100 µg/mL kanamycin. Incubate in a 37 °C shaking incubator at 120 rpm for 2 h, and then, add the inducer lactose to a final concentration of 0.5 mmol/L and continue to shake for 6 h, inducing EGFP expression. Empirically, when shaking for 6 h, the optical density at 600 nm (OD600) may reach 0.7 or higher.
7. Add 10 µL of the bacterial culture medium onto a slide, cover it with a coverslip, and examine the expression of EGFP under an inverted fluorescence microscope. The EGFP-expressing bacteria can be stored in the medium at 2-8 °C for several weeks.

2. Mouse peritoneal macrophage isolation and primary culture

1. Add 3.5 g of thioglycolate to 100 mL of distilled water and autoclave the mixture to sterility before use. Pump the thioglycolate medium into the 1 mL sterile syringe in the hood for the mouse peritoneal injection. Use one mouse per syringe to avoid infection. The use of thioglycolate can increase the number of macrophages. The resident peritoneal macrophages can be isolated without thioglycolate but with lower macrophage yields.
2. Anesthetize the mouse using a method approved by the local animal care and use committee. Inject 1 mL of 3.5% thioglycolate medium into the mouse's peritoneal cavity with the 1 mL syringe, using a 23 G needle.
   NOTE: By inducing anesthesia, the peritoneal injection can be easily performed and reduces the risk of injuries to the internal organs caused by injection.
3. Maintain the mouse with water and food ad libitum for 3 days. Monitor the body weight and food intake of the animal every day. If the body weight loss is greater than 10% within 3 days, exclude the animal from the experiment.
4. After 3 days, euthanize the mouse by cervical dislocation after rapidly inducing anesthesia by sevoflurane in a closed box. Alternatively, use a method that has been approved by the local animal care and use committee to euthanize the mouse.
5. Put the mouse into a dish (with a 10 cm diameter) with 75% ethanol to sterilize, and transfer it quickly to the hood. Place the mouse on a plate and pin the front paw to the board to fix the mouse's position.
6. Using a 5 mL syringe attached to a 20 G needle, placing the needle bevel up at a 30°-40° angle, inject 5 mL of cold (4-10 °C) phosphate-buffered saline (PBS) at the lower abdomen into the mouse's peritoneal cavity, avoiding puncturing the bowel. If the bowel (or any other organ) is punctured, the mouse and its cells can no longer be used for experiments, as this may activate cells that are not suitable for primary cell culture.
7. Perform a gentle massage on the two sides of the mouse's abdomen. Then, aspirate the fluid gently and slowly. Dispense the peritoneal fluid into a 50 mL centrifuge tube. Repeat these steps 2x or 3x.
8. Centrifuge the suspended cells for 10 min at 400 x g in a refrigerated centrifuge (4-8 °C). Discard the supernatant and resuspend the cell pellet in RPMI 1640 medium with 10% fetal bovine serum (FBS). Count the cells. Empirically, the cell density is approximately equal to 5 x 10⁶ cells/mL when the cells are resuspended in 10 mL of medium.
9. Add 5 x 10⁶ cells into each well of a 6-well plate for the flow cytometry assay and 5 x 10⁵ cells per well into a 24-well plate for a fluorescence microscope. Culture the cells at 37 °C in a 5% CO₂ incubator overnight. The culture medium can be refreshed after 3 h to remove nonadherent cells because most of these are lymphocytes. The adherent cells are mainly the macrophages, and they can adhere well to tissue-culture-treated plastic.

3. Macrophage phagocytosis assay using the fluorescence microscope

1. Observe the cells under a bright-field microscope to evaluate cell viability and cell density.
2. Remove the culture medium from the 24-well plate. Add 100 µL of fresh culture medium and 10 µL of bacterial suspension (approximately 2 x 10⁷ cells) into each well as described in Table 1. Incubate for 1 h in a 37 °C, 5% CO₂ incubator.
3. Gently wash 3x-5x with 500 µL of cold PBS per well to wash out noninternalized bacteria.
4. Incubate the cells with 4% formaldehyde in PBS at room temperature for 30 min.
5. Wash the fixed cells 3x with PBS (500 µL/well).
6. Add 200 µL of phalloidin 633 fluorescence dye conjugated working solution (see Table of Materials) to stain the F-actin. Store in a dark, humid place (60%-80%) at room temperature for 60 min. Rinse the cells 3x with PBS (500 µL/well) to remove any excess phalloidin. By staining the F-actin, the cytoplasm can be outlined and help to distinguish the internalized bacteria.
7. Add 200 µL of DAPI (4',6-diamidino-2-phenylindole) working solution (1 µg/mL) to stain the cell nucleus and incubate for 5 min in a dark, humid place at room temperature. Rinse 1x with PBS (500 µL/well) and 1x with the same volume of distilled water. Then, the cells will be ready for observation under an inverted fluorescence microscope.

4. Macrophage phagocytosis assay using flow cytometry

1. To minimize the experimental errors and make a proper interpretation of the results, set the groups and control tubes for the experiment as listed in Table 2.
   1. For the control group, which will be placed on ice (Group 4 in Table 2), remove the medium from the 6-well plate and wash it 1x with PBS. Then, add 1 mL of 70 mM cold EDTA into the well to detach the cells and transfer them to the flow cytometry tube. Add 50 µL of bacterial suspension into the tube and place it on ice for 1 h.
   2. For the other groups, remove the culture medium. Add 1 mL of fresh medium into each well. Add 50 µL of bacterial suspension into the wells according to the group setting, as described in Table 2. Then, place the 6-well plate in the 37 °C, 5% CO₂ incubator for 1 h.
2. To quench the fluorescence of noninternalized E. coli, add 200 µL of 0.8% crystal violet (CV) water solution into the well and sway shortly, thus avoiding a false-positive result by the EGFP-expressing E. coli binding to the surface of the macrophages but not internalized. Wash the cells 3x with PBS to remove any residual CV.
3. Then, add 1 mL of 70 mM cold EDTA into the well to detach the cells and transfer them to the flow cytometry tube.
4. Centrifuge the tubes at 400 x g for 5 min and discard the supernatant.
5. Add 100 µL of PBS to resuspend the cells. Add 5 µL of F4/80-PE-conjugated antibody (a surface antigen expressed on mouse macrophages) into the tubes, or use IgG2a-PE isotype, according to the group setting. Vortex briefly and incubate the samples on ice for 5-10 min in the dark.
6. Add 1 mL of PBS into each tube and centrifuge at 400 x g for 5 min. Discard the supernatant. Resuspend the cell pellets with 200-300 µL of PBS for flow cytometry analysis. Run each tube and acquire data for at least 10,000 events of F4/80⁺ cells.

Representative Results

http://jovecomapi-svc-main-service/api/free/article/de/58751

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

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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

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