The National Natural Science Foundation of China (no. 31800046) and the Natural Science Foundation of Liaoning Province (no. 20170540262) supported this work. This work was accomplished in the laboratories of the Scientific Research Center at the Second Hospital of Dalian Medical University. The authors would like to thank Xiao-Lin Sang for her assistance with the flow cytometry, and Bo Qu and Dong-Chuan Yang for their assistance in producing the video.

&#8203;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 &#176;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
<ol>
	<li>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&#39;-ATGGTGAGCAAGGGCGAGGAGC-3&#39;) and reverse primer (5&#39;-CTTGTACAGCTCGTCCATGCCG-3&#39;), using high-fidelity Taq DNA polymerase.</li>
	<li>To ensure that the polymerase chain reaction (PCR) products have single 3&#39; adenine overhangs for the TA cloning in the next step, use a 30 min extension at 72 &#176;C after the last cycle (see Supplementary File 1 for PCR conditions). Check the PCR product by agarose gel electrophoresis.</li>
	<li>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 &#176;C) for 30 min. The vector is linearized between nucleotides 653 and 654 with a 1 bp 5&#8242; T-overhang on each strand.</li>
	<li>Transform the ligation product into the chemically competent BL21(DE) E. coli strain as follows: add 5 &#181;L (100 ng) of PCR product to 100 &#181;L of BL21(DE) competent cells via heat shock at 42 &#176;C for 90 s; keep the mixture on ice for 3 min, and then, add 400 &#181;L of lysogeny broth (LB) medium preheated at 37 &#176;C, shaking it for 1 h at 37 &#176;C and 120 rpm.</li>
	<li>Inoculate 100 &#181;L of the bacteria onto the surface of an LB-kanamycin (100 &#181;g/mL) plate with the inducer lactose (0.5 mmol/L), yielding the EGFP expression strain. Incubate the plate at 37 &#176;C overnight. 
	NOTE: If the EGFP is successfully expressed, some colonies can be observed as glowing green light in the dark.
	<ol>
		<li>Optionally, select colonies to verify the inserted EGFP fragment by DNA sequencing. The primers for DNA sequencing are: forward, 5&#39;-AGATTCTTGTACGACGGTATTAG-3&#39;; reverse, 5&#39;-TAGTTATTGCTCAGCGGTGG-3&#39;.</li>
	</ol>
	</li>
	<li>Inoculate a positive colony into 5 mL of LB medium with 100 &#181;g/mL kanamycin. Incubate in a 37 &#176;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.</li>
	<li>Add 10 &#181;L 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 &#176;C for several weeks.</li>
</ol>
2. Mouse peritoneal macrophage isolation and primary culture
<ol>
	<li>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.</li>
	<li>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&#39;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#39;s position.</li>
	<li>Using a 5 mL syringe attached to a 20 G needle, placing the needle bevel up at a 30&#176;-40&#176; angle, inject 5 mL of cold (4-10 &#176;C) phosphate-buffered saline (PBS) at the lower abdomen into the mouse&#39;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.</li>
	<li>Perform a gentle massage on the two sides of the mouse&#39;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.</li>
	<li>Centrifuge the suspended cells for 10 min at 400 x g in a refrigerated centrifuge (4-8 &#176;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.</li>
	<li>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 &#176;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.</li>
</ol>
3. Macrophage phagocytosis assay using the fluorescence microscope
<ol>
	<li>Observe the cells under a bright-field microscope to evaluate cell viability and cell density.</li>
	<li>Remove the culture medium from the 24-well plate. Add 100 &#181;L of fresh culture medium and 10 &#181;L of bacterial suspension (approximately 2 x 107 cells) into each well as described in Table 1. Incubate for 1 h in a 37 &#176;C, 5% CO2 incubator.</li>
	<li>Gently wash 3x-5x with 500 &#181;L of cold PBS per well to wash out noninternalized bacteria.</li>
	<li>Incubate the cells with 4% formaldehyde in PBS at room temperature for 30 min.</li>
	<li>Wash the fixed cells 3x with PBS (500 &#181;L/well).</li>
	<li>Add 200 &#181;L 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 &#181;L/well) to remove any excess phalloidin. By staining the F-actin, the cytoplasm can be outlined and help to distinguish the internalized bacteria.</li>
	<li>Add 200 &#181;L of DAPI (4&#39;,6-diamidino-2-phenylindole) working solution (1 &#181;g/mL) to stain the cell nucleus and incubate for 5 min in a dark, humid place at room temperature. Rinse 1x with PBS (500 &#181;L/well) and 1x with the same volume of distilled water. Then, the cells will be ready for observation under an inverted fluorescence microscope.</li>
</ol>
4. Macrophage phagocytosis assay using flow cytometry
<ol>
	<li>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.
	<ol>
		<li>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 &#181;L of bacterial suspension into the tube and place it on ice for 1 h.</li>
		<li>For the other groups, remove the culture medium. Add 1 mL of fresh medium into each well. Add 50 &#181;L 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 &#176;C, 5% CO2 incubator for 1 h.</li>
	</ol>
	</li>
	<li>To quench the fluorescence of noninternalized E. coli, add 200 &#181;L 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.</li>
	<li>Then, add 1 mL of 70 mM cold EDTA into the well to detach the cells and transfer them to the flow cytometry tube.</li>
	<li>Centrifuge the tubes at 400 x g for 5 min and discard the supernatant.</li>
	<li>Add 100 &#181;L of PBS to resuspend the cells. Add 5 &#181;L 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.</li>
	<li>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 &#181;L of PBS for flow cytometry analysis. Run each tube and acquire data for at least 10,000 events of F4/80+ cells.</li>
</ol>

<ol>
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The authors have nothing to disclose.

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 light signal was strong enough to be detected by both a fluorescence microscope and a flow cytometer. 
The other critical step is to quench the fluorescence of bacteria which were not internalized by the macrophages. Although Trypan Blue has been shown to quench the fluorescence of fluorescein isothiocyanate (FITC)-labeled, heat-killed bacteria, it did not work for the live E. coli. Using a 0.8% crystal violet water solution can quench most of the fluorescence of the E. coli which bind on the cell surface. Some literature suggests that washing with antibiotics instead of with Trypan Blue may help to quench the fluorescence, but that was not effective in this experiment10.
The cell density may limit this technique. Because the cells consist of a mixture of lymphocytes and macrophages, the macrophages are usually lower than the cell density calculated from the hemocytometer when harvesting the cells from the mouse peritoneal cavity, which may result in an insufficient number of cells for the flow cytometry and the fluorescence microscopy. In the case of insufficient numbers of macrophages, cells from two to three mice within the same group may mix for the phagocytosis assay. When this technique is applied to macrophage cell lines, such as RAW264.7, cell loss may be a concern, because these cells are relatively nonadherent; thus, cells may be lost during the washing procedure. Wash gently or use culture plates with cell-treated surfaces, which may increase the cell adhesion.
There are many other methods to assess phagocytosis ability. As one of the classic methods, chicken erythrocytes or stained dead cells were used as markers of phagocytosis. The sensitivity of these methods was limited by the considerable variation of the results. Another alternative method for examining phagocytosis is to use cells infected with bacteria for several hours, then lyse the cells with Triton X-100 and plate on an LB agar Petri dish overnight at 37 &#176;C. The phagocytic capacity is determined by counting the number of colony-forming units (CFUs)6. This method required as long as 2 days to obtain the CFU data, and the variance of the counted numbers was large because the cell lysates are diluted several times. Then, FITC-labelled beads7 or E. coli were introduced for the phagocytosis assays8. Because these beads lacked specific surface antigens, additional preopsonization was required for optimal uptake. Also, the method of using the FITC-labeled bacteria might hinder the phagocytosis because the FITC compromised the bacterial virulence9.
Another newly introduced method is to use commercialized dyes, which are pH sensitive and only fluoresce once they are inside the acidic lysosome, thus eliminating the quenching step10. However, the commercialized kit may be cost prohibitive. Once the EGFP-expressing E. coli strain is constructed, the bacteria are easily reproduced, and the fluorescence is stable for several weeks, which makes this method simple and economical. Because the EGFP has a strong fluorescence, this method can also be modifiable to a high-throughput fluorometric technique to assess macrophage phagocytosis, which can be performed in an opaque 96-well plate11.

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 body2,3. 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.

<table border="0" cellpadding="0" cellspacing="0" style="width:886px;" width="887">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:417px;">BD FACSCanto II Flow cytometer</td>
			<td style="width:176px;">BD Biosciences</td>
			<td style="width:123px;">-</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:417px;">Biotin anti-mouse CD16/32 Antibody</td>
			<td style="width:176px;">Biolegend</td>
			<td style="width:123px;">Cat101303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:417px;">Champion pET SUMO Protein Expression system</td>
			<td style="width:176px;">Invitrogen</td>
			<td style="width:123px;">K300-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:417px;">Custom Gene Synthesis Service</td>
			<td style="width:176px;">Takara Biotech.</td>
			<td style="width:123px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:417px;">DAPI(4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:176px;">ThermoFisher</td>
			<td style="width:123px;">D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:417px;">F4/80-PE anti-mouse antibody for FACS</td>
			<td style="width:176px;">Biolegend</td>
			<td style="width:123px;">Cat123110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:417px;">Leica DMI3000 B&#160; Inverted Microscope</td>
			<td style="width:176px;">Leica Microsystems</td>
			<td style="width:123px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:417px;">PE Rat IgG2a, &#954;-isotype control</td>
			<td style="width:176px;">Biolegend</td>
			<td style="width:123px;">Cat400507</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:417px;">Phalloidin 633 fluorescence dye conjugated working solution</td>
			<td style="width:176px;">AAT Bioquest</td>
			<td style="width:123px;">Cat23125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:417px;">Thioglycollate medium</td>
			<td style="width:176px;">Sigma-Aldrich</td>
			<td style="width:123px;">T9032</td>
			<td></td>
		</tr>
	</tbody>
</table>

using enhanced green fluorescence protein-expressing escherichia coli to assess mouse peritoneal macrophage phagocytosis

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 &#176;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.

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 objective lens (Figure 1B).
Microscopy analysis shows fluorescence images (Figure 1C) of peritoneal macrophages from the young and aged groups. Figure 1C shows the red fluorescence of F-actin, the green fluorescence of EGFP-expressing E. coli, the blue fluorescence of DAPI nuclear staining, and the merged image of all three fluorescence channels. The 16-month-old mice, which were regarded as the aged mice, were equivalent of 60- to 65-year-old humans. These images suggest that macrophages from the young mice presented a stronger phagocytosis ability than those from the aged mice.
Flow cytometry (Figure 2) was used to quantify and compare macrophage phagocytosis from the young and aged group. Figure 2A shows a representative flow cytometry analysis of the young, aged, and control groups. The F4/80-PE antibody was used to identify and gate the macrophages, and EGFP-positive signals indicate the macrophages that phagocytosed E. coli. The proportion of F4/80+ and EGFP+ cells indicate the phagocytic ability of the macrophages. The result (Figure 2B) of the young group was 62.7% &#177; 5.1% (mean &#177; SEM), which was significantly higher than the 35.2% &#177; 2.9% (mean &#177; SEM) of the aged group. These results are consistent with the trend of fluorescence microscopy results.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58751/58751fig1.jpg" /> 
Figure 1: EGFP-expressing E. coli and its phagocytosis by macrophages. (A) EGFP-expressing E. coli colonies. The pET-SUMO-EGFP plasmid was transformed into BL21(DE) cells; the bacteria were inoculated on an LB-kanamycin (100 &#181;g/mL) plate. A coating of 0.5 mmol/L lactose on the LB plate surface was used as the inducer, yielding the EGFP expression. If the EGFP is expressed successfully, yellowish green colonies are observed using UV light in the dark. (B) Fluorescence microscopy of EGFP-expressing E. coli. The green signal represents EGFP-expressing E. coli. Scale bar = 50 &#181;m. (C) Multichannel fluorescence images of macrophages that were phagocytosing E. coli. The cells were incubated with EGFP-expressing E. coli (green) for 1 h, followed by a wash with PBS, fixation with 4% paraformaldehyde, and staining for F-actin using phalloidin 633 conjugate working solution (red) and DAPI (blue). Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58751/58751fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58751/58751fig2.jpg" /> 
Figure 2: Flow cytometry results. (A) Representative flow cytometry analysis of the young, aged, and control groups. The peritoneal macrophages were stained with F4/80-PE after coincubation with EGFP-expressing E. coli. F4/80+ and EGFP+ cells were rare in the negative control and control (group 4: young group on ice) groups. The young and aged flow cytometric plots represent groups 5 and 6, respectively. (B) The results from the flow cytometry analysis of the young and aged groups. A Mann-Whitney test was used to examine the difference between these two groups. The proportion of F4/80+ and EGFP+ cells in the young group was significantly higher than that in the aged group (*P &#60; 0.05). The error bars represent the standard error of the mean (SEM). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58751/58751fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Group</td>
			<td>Name</td>
			<td>Cells</td>
			<td>EGFP E. coli</td>
			<td>Co-incubation time</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Young</td>
			<td>2 x 105</td>
			<td>2 x 107</td>
			<td>1 h</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Aged</td>
			<td>2 x 105</td>
			<td>2 x 107</td>
			<td>1 h</td>
		</tr>
	</tbody>
</table>
Table 1: Group setting for fluorescence microscopy. Two groups, the aged group (16-month-old C57BL/6, n = 3) and the young group (8-week-old C57BL/6, n = 3), were used to prepare peritoneal macrophages. The peritoneal macrophages of each mouse were added to separate wells. Approximately 2 x 105 cells in a volume of 100 &#181;L were added to each well; then, approximately 2 x 107 EGFP-expressing E. coli cells in a volume of 10 &#181;L were added to each well and coincubated for 1 h at 37 &#176;C.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Group</td>
			<td rowspan="2">Name and condition</td>
			<td rowspan="2">Cells</td>
			<td>EGFP&#160;</td>
			<td rowspan="2">F4/80-PE</td>
			<td rowspan="2">PE ISOTYPE</td>
		</tr>
		<tr>
			<td>E. coli</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Isotype control at 37&#176;C</td>
			<td>2 x 106</td>
			<td>&#8212;</td>
			<td>&#8212;</td>
			<td>Add 5 &#956;L</td>
		</tr>
		<tr>
			<td>2</td>
			<td>PE Positive control at 37&#176;C</td>
			<td>2 x 106</td>
			<td>&#8212;</td>
			<td>Add 5 &#956;L</td>
			<td>&#8212;</td>
		</tr>
		<tr>
			<td>3</td>
			<td>EGFP Positive control at 37&#176;C</td>
			<td>2 x 106</td>
			<td>1 x 108</td>
			<td>&#8212;</td>
			<td>&#8212;</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Young group on ice</td>
			<td>2 x 106</td>
			<td>1 x 108</td>
			<td>Add 5 &#956;L</td>
			<td>&#8212;</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Young group at 37&#176;C</td>
			<td>2 x 106</td>
			<td>1 x 108</td>
			<td>Add 5 &#956;L</td>
			<td>&#8212;</td>
		</tr>
		<tr>
			<td>6</td>
			<td>Age group at 37&#176;C</td>
			<td>2 x 106</td>
			<td>1 x 108</td>
			<td>Add 5 &#956;L</td>
			<td>&#8212;</td>
		</tr>
	</tbody>
</table>
Table 2: Group setting for flow cytometry. The primary peritoneal macrophages from the young and aged mice were set as six groups. Group 1 was set as isotype control; groups 2 and 3 were set as single positive control for the PE or EGFP channel, respectively. To ensure that the internalized fluorescence is specific to the phagocytosis, group 4 was incubated on ice. The phagocytosis is stopped on ice because of the low temperature. The incubation time was 1 h for all the groups.

Watch this Scientific Journal Video about Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis at JoVE.com

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

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

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

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...

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Unit 1

Endocytosis and Exocytosis

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7.3K Views.  The Second Hospital of Dalian Medical University. Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

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

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established.
In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin.
This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

A high yield method for one-step negative purification of recombinant Helicobacter pylori neutrophil-activating protein (HP-NAP) overexpressed in Escherichia coli by using diethylaminoethyl resins in batch mode is described. HP-NAP purified by this method is beneficial for the development of vaccines, drugs, or diagnostics for H. pylori-associated diseases.

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

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

Summary

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Chapters in this video

Isolation of Mouse Peritoneal Cavity Cells

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli