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>
	<li>Layoun, A., Samba, M., Santos, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+murine+peritoneal+macrophages+to+carry+out+gene+expression+analysis+upon+Toll-like+receptors+stimulation.">Isolation of murine peritoneal macrophages to carry out gene expression analysis upon Toll-like receptors stimulation.</a> Journal of Visualized Experiments. 98, e52749 (2015).</li><li>Iskander, K. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sepsis:+multiple+abnormalities,+heterogeneous+responses,+and+evolving+understanding.">Sepsis: multiple abnormalities, heterogeneous responses, and evolving understanding.</a> Physiological Reviews. 93 (3), 1247-1288 (2013).</li><li>Girard, T. D., Opal, S. M., Ely, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+severe+sepsis+in+older+patients:+from+epidemiology+to+evidence-based+management.">Insights into severe sepsis in older patients: from epidemiology to evidence-based management.</a> Clinical Infectious Diseases. 40 (5), 719-727 (2005).</li><li>Bicker, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+assay+to+measure+phagocytosis+of+live+bacteria.">A simple assay to measure phagocytosis of live bacteria.</a> Clinical Chemistry. 54 (5), 911-915 (2008).</li><li>Zhou, M. Y., Gomez-Sanchez, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+TA+cloning.">Universal TA cloning.</a> Current Issues in Molecular Biology. 2 (1), 1-7 (2000).</li><li>Chen, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triggering+receptor+expressed+on+myeloid+cells-2+protects+against+polymicrobial+sepsis+by+enhancing+bacterial+clearance.">Triggering receptor expressed on myeloid cells-2 protects against polymicrobial sepsis by enhancing bacterial clearance.</a> American Journal of Respiratory and Critical Care Medicine. 188 (2), 201-212 (2013).</li><li>Lamberti, G., de Araujo, M. E., Huber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Macrophage+Early+and+Late+Endosomes+by+Latex+Bead+Internalization+and+Density+Gradient+Centrifugation.">Isolation of Macrophage Early and Late Endosomes by Latex Bead Internalization and Density Gradient Centrifugation.</a> Cold Spring Harbor Protocols. (12), (2015).</li><li>Miksa, M., Komura, H., Wu, R., Shah, K. G., Wang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+to+determine+the+engulfment+of+apoptotic+cells+by+macrophages+using+pHrodo+succinimidyl+ester.">A novel method to determine the engulfment of apoptotic cells by macrophages using pHrodo succinimidyl ester.</a> Journal of Immunological Methods. 342 (1-2), 71-77 (2009).</li><li>Weingart, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+labels+influence+phagocytosis+of+Bordetella+pertussis+by+human+neutrophils.">Fluorescent labels influence phagocytosis of Bordetella pertussis by human neutrophils.</a> Infection and Immunity. 67 (8), 4264-4267 (1999).</li><li>Neaga, A., Lefor, J., Lich, K. E., Liparoto, S. F., Xiao, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+flow+cytometric+method+to+evaluate+phagocytosis+of+pHrodo+BioParticles(R)+by+granulocytes+in+multiple+species.">Development and validation of a flow cytometric method to evaluate phagocytosis of pHrodo BioParticles(R) by granulocytes in multiple species.</a> Journal of Immunological Methods. 390 (1-2), 9-17 (2013).</li><li>Ninkovic, J., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+fluorometric+technique+for+assessment+of+macrophage+phagocytosis+and+actin+polymerization.">High throughput fluorometric technique for assessment of macrophage phagocytosis and actin polymerization.</a> Journal of Visualized Experiments. (93), e52195 (2014).</li></ol>

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 ...

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><tbody><tr><td>BD FACSCanto II Flow cytometer</td><td>BD Biosciences</td><td>-</td><td></td></tr><tr><td>Biotin anti-mouse CD16/32 Antibody</td><td>Biolegend</td><td>Cat101303</td><td></td></tr><tr><td>Champion pET SUMO Protein Expression system</td><td>Invitrogen</td><td>K300-01</td><td></td></tr><tr><td>Custom Gene Synthesis Service</td><td>Takara Biotech.</td><td>-</td><td></td></tr><tr><td>DAPI(4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>ThermoFisher</td><td>D1306</td><td></td></tr><tr><td>F4/80-PE anti-mouse antibody for FACS</td><td>Biolegend</td><td>Cat123110</td><td></td></tr><tr><td>Leica DMI3000 B&amp;nbsp; Inverted Microscope</td><td>Leica Microsystems</td><td>-</td><td></td></tr><tr><td>PE Rat IgG2a, &amp;kappa;-isotype control</td><td>Biolegend</td><td>Cat400507</td><td></td></tr><tr><td>Phalloidin 633 fluorescence dye conjugated working solution</td><td>AAT Bioquest</td><td>Cat23125</td><td></td></tr><tr><td>Thioglycollate medium</td><td>Sigma-Aldrich</td><td>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...

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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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

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 ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the removal of dead cells or foreign particles, and in the resolution of inflammatory responses and tissue remodeling, processes that are mediated by various surface receptors of the AMs. Here, we report methods for the analysis of the phagocytic function of AMs using in vitro and in vivo assays and experimental strategies to differentiate between the pattern recognition receptor-, complement receptor-, and Fc gamma receptor-mediated phagocytosis. Finally, we discuss a method to establish and characterize a P. aeruginosa pneumonia model in mice to assess bacterial clearance in vivo. These assays represent the most common methods to evaluate AM functions and can also be used to study macrophage function and bacterial clearance in other organs.

Here we report common methods to analyze the phagocytic function of murine alveolar macrophages and bacterial clearance from the lung. These methods study in vitro phagocytosis of fluorescein isothiocyanate beads and in vivo phagocytosis of Pseudomonas aeruginosa Green Fluorescent Protein. We also describe a method for clearing P. aeruginosa in mice.

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Medicine

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...

Phagocytic Assay Evaluation of Neonatal Bone Marrow Derived Macrophages 

Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging and time-consuming, yet for some models, it is translationally relevant and necessary. This protocol describes an efficient and straightforward method for preparing bone marrow cells from 7-9-day-old pups. These cells can then be further isolated or differentiated into specific cell types of interest. Macrophages are crucial immune cells that play a major role in inflammation and infection. During development, neonatal macrophages contribute significantly to tissue remodeling. Moreover, the phenotype and functions of neonatal macrophages differ from those of their adult counterparts. This protocol also outlines the differentiation of neonatal macrophages from the isolated bone marrow cells in the presence of L929-conditioned medium. Surface markers for differentiated neonatal macrophages were assessed using flow cytometric analysis. To demonstrate functionality, the phagocytic efficiency was also tested using pH-sensitive dye-conjugated Escherichia coli.

Author Spotlight: Developing a Reproducible Method for Isolating Bone Marrow-Derived Macrophages from Neonatal Mice to Advance Early Life Immune Responses

This protocol describes a non-enzymatic and straightforward method for isolating 7-9-day-old neonatal mouse bone marrow cells and generating differentiated macrophages using a supernatant of L929 cells as a source of granulocyte colony-stimulating factor (M-CSF). The bone marrow-derived macrophages were further analyzed for surface antigens F4/80, CD206, CD11b, and functional competency.

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging ...

Using Fluorescence-Mediated Tomography to Assess Murine Intestinal Inflammation

Source: Nowacki, T. M., et al. <a href="https://app.jove.com/v/55942">Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation.</a> J. Vis. Exp. (2017)
This video demonstrates a technique for assessing intestinal macrophage infiltration in a murine model of colitis using fluorescence-mediated tomography (FMT). Upon inducing colitis in a mouse via dextran sodium sulfate (DSS), the animal is injected with antibodies that recognize the F4/80 glycoprotein marker on infiltrated macrophages in the colon. The mouse is then imaged in an FMT device where the fluorescence and reflectance signals are reconstructed into a three-dimensional image to determine the spatial distribution and intensity of fluorescence.

Wykorzystanie tomografii fluorescencyjnej do oceny zapalenia jelit myszy

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

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Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages

Wykorzystanie tomografii fluorescencyjnej do oceny zapalenia jelit myszy