Research in the Shao Lab is supported by Research Innovative Award from the College of Medicine and the Junior Faculty Pilot Award from the Department of Internal Medicine, University of Cincinnati and grant DK K01_095067 from NIDDK/NIH.

Experimental mice were bred and maintained in our mice colony. All animal work was conducted according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the University of Cincinnati.
1. Preparation of CFSE labeled apoptotic thymocytes
<ol>
	<li>Euthanize two na&#239;ve C57/B6 mice by CO2 inhalation for 10 min and dissect to open the chest cavity, remove (pull out) the thymus with curved fine-tip forceps into tissue culture petri dish containing 10 mL of RPMI1640 medium.</li>
	<li>Obtain single cell suspension by grinding the whole thymus against two frosted ends of the microscope slides and then filter the suspension through 100 &#956;m cell strainer.</li>
	<li>Collect the 10 mL thymus suspension into a 50 mL tube and centrifuge at 300 x g for 5 min.</li>
	<li>Remove the supernatant, resuspend in 40 mL of 1x PBS, and count cell numbers with a hemocytometer.</li>
	<li>Centrifuge at 300 x g for 5 min and remove the supernatant.</li>
	<li>Resuspend in 20 mL of 1x PBS in a 50 mL tube as a single cell suspension with up to 2 x&#160;108 cells (if more than 2 x&#160;108 cells, resuspend the rest of the cells in another 20 mL of 1 x&#160;PBS in a different 50 mL tube).</li>
	<li>Make 5 &#956;M of CFSE in equal volume (20 mL) of 1x PBS in a separate 50 mL tube by pipetting 40 &#956;L of CFSE stock solution (2.5 mM) into 20 mL of 1x PBS and mix well by inverting the tube 2 - 3 times.</li>
	<li>Add the 20 mL of CFSE from step 1.7 into the 20 mL of cell suspension from step 1.6 (the final concentration of CFSE is now 2.5 &#956;M). 
	NOTE: For every 20 mL cell suspension, a 20 mL of CFSE tube is needed.</li>
	<li>Invert the cell and CFSE mixture tube 2 - 3 times and incubate the mixture in the dark at room temperature for a maximum of 2 min, then stop the reaction by adding 10 mL of heat-inactivated horse serum.</li>
	<li>Centrifuge the 50 mL tube mixture at 300 x g for 5 min at room temperature. 
	NOTE: If the CFSE labeling is successful, cell pellet will become light yellow in color.</li>
	<li>Remove the supernatant and resuspend the cell pellet in 40 mL of 1x PBS and count cell numbers with a hemocytometer.</li>
	<li>Centrifuge the cell suspension at 300 x g for 5 min and discard the supernatant.</li>
	<li>Wash the cell pellet again with 40 mL of RPMI1640 medium.</li>
	<li>Remove the supernatant and resuspend cells with RPMI1640 tissue culture medium (RPMI1640, 20 mM HEPES, 10% FBS (heat inactivated), 20 mM glutamine, and 1x Pen/Strep) at a concentration of 7 x&#160;106 cells/mL in a 100 mm tissue culture dish. 
	NOTE: If more cells are obtained, a separate 100 mm tissue culture dish will be needed.</li>
	<li>Add staurosporine into the cell suspension culture at a final concentration of 1 &#956;M and culture for 4 h at 37 &#176;C in a tissue culture incubator supplied with 5% CO2.</li>
</ol>
2. Preparation of peritoneal macrophages
<ol>
	<li>Inject two C57B6 mice (or any gene-manipulated mice in the lab) intraperitoneally with 1 mL of 3% aged thioglycollate at day 0.</li>
	<li>Euthanize the mice at day 5 as in step 1.1, cut and peel open the abdominal skin but leave the peritoneum intact. Flush the peritoneal cavity by quickly pushing 10 mL of wash buffer (RPMI1640, 2% FBS, 0.04% EDTA) into the peritoneal cavity using a 10 mL syringe attached with a 18&#160;G needle.</li>
	<li>Retrieve the wash buffer slowly with the same needle/syringe, and collect the wash buffer into a 50 mL tube (details refer to Janssen lab article15).</li>
	<li>Wash the peritoneal gavage twice with 1x PBS, resuspend the peritoneal macrophages in RPMI1640 tissue culture medium at a density of 2 x&#160;106 cells/mL and aliquot 500 &#956;L into each well of the 24-well plate. Leave the plate in a tissue culture incubator for 2 h.</li>
	<li>Remove the floating cells by aspirating and replacing with 500 &#956;L of fresh culture medium, twice.</li>
	<li>Optionally, add TAM receptor tyrosine inhibitor, RXDX-106, at concentrations indicated in the figure legends into each well of the macrophage culture and incubate for another 2 h.</li>
</ol>
3. Co-culture of peritoneal macrophages with apoptotic thymocytes
<ol>
	<li>Collect apoptotic thymocytes from step 1 and wash three times with RPMI1640 medium. The efficiency of apoptotic induction can be measured at this stage with the Annexin V/7-AAD kit.</li>
	<li>Distribute 0 - 12 x&#160;106 cells (in 500 &#956;L medium) into each well of the macrophage cultures from protocol #2, according to the experimental arrangement (e.g., see Figure 1). This makes the whole culture volume of 1 mL in each well of the 24-well plate. Add the blocking antibody into the culture immediately before the addition of apoptotic thymocytes.</li>
	<li>Culture the cell mixture at 37 &#176;C for 4 h in a tissue culture incubator supplied with 5% CO2.</li>
	<li>Wash each well of the culture with 1x PBS (containing 500 &#956;M EDTA) twice to remove the free-floating apoptotic cells.</li>
	<li>Stain the plate-bound macrophages with CD11b-PE at this stage.
	<ol>
		<li>Wash the plate-bound macrophages with staining buffer (1x PBS, 1% BSA) once.</li>
		<li>Add 200 &#956;L of staining buffer containing 2 &#956;L CD11b-PE into each well.</li>
		<li>Incubate the plate at 4 &#176;C for 20 min.</li>
		<li>Wash each well of plate three times with the staining buffer.</li>
		<li>Add 200 &#956;L of staining buffer and proceed the plate for image analysis under the fluorescent microscope.</li>
	</ol>
	</li>
	<li>Alternatively, detach the plate-bound macrophages by adding 1 mL of 1% lidocaine in PBS and incubating for 10 min at 37 &#176;C.</li>
	<li>Detach plate-bound macrophages with repeat pipetting.</li>
	<li>Transfer macrophage suspension into an individual 5 mL round-bottom FACS tube from each well of the 24-well plate.</li>
	<li>Centrifuge at 300 x g for 5 min.</li>
	<li>Remove the supernatant and add 200 &#956;L of staining buffer containing 2 &#956;L of CD11b-PE (1:200 dilution in staining buffer).</li>
	<li>Incubate the cell suspension in staining buffer at 4 &#176;C for 20 min.</li>
	<li>Wash the cell suspension twice with staining buffer.</li>
	<li>Add 200 &#956;L of staining buffer and proceed for FACS analysis with a flow cytometer and analyze for the percentage of CFSE positivity macrophages.</li>
</ol>

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The authors have nothing to disclose.

Apoptosis is a highly conserved cell death process that involves many signal cascades and induces protein expression, secretion, and transportation. Apoptosis is often associated with cellular morphology changes17. Apoptotic cells actively release cytokines and chemokines that attract phagocytes to migrate to the site and initiate the process of engulfment, an extremely complex pathway under tight control18. On the other hand, necrotic cell death releases danger signals tha...

Our body generates 1-10 billion apoptotic cells on a daily basis. Such a large number of apoptotic cells must be cleared in a way that the immune responses remain quiescent. To ensure the clearance of apoptotic cells in a timely manner, numerous types of tissue resident cells and circulating cells develop mechanisms to engulf apoptotic cells1. Dysfunctional regulation of apoptosis has been implicated in the onset and progression of various inflammatory disease and autoimmunity2. Apoptosis also plays a critical role in the pathogenesis of cancer development and its subsequent resistance to conventional treatments3,4. Removal of apoptotic cells generally promotes an anti-inflammatory response, which may be linked to immunological tolerance5. Disturbance of apoptotic cell clearance drives self-immunization and contributes to the development of systemic autoimmune diseases in both humans and mice6.
When cells undergo apoptosis, they expose the phosphatidylserine (PtdSer) from the inner leaflet to the outer leaflet of the membrane. PtdSer will then be recognized by phagocytes through surface receptors. Over a dozen receptors have been identified to recognize and/or facilitate the engulfment of apoptotic cells. In general, there are at least three types of surface receptors involved in the apoptotic cell clearance: tethering receptors, recognize apoptotic cells; tickling receptors, initiate engulfment; chaperoning receptors, facilitate the whole process7. TAM receptor tyrosine kinases (TAM RTKs) consist of Tyro-3, Axl, and Mer and are primarily expressed by myeloid cells of the immune system8. The primary function of TAM RTKs is to serve as tethering receptors, facilitating the phagocytic removal of apoptotic cells and debris. Our group has studied TAM mediated apoptotic cell clearance in the setting of autoimmunity for many years. The vitamin K-dependent protein growth arrest specific protein 6 (Gas6) and protein S (ProS) binds to and activates TAM receptors9,10. Gas6 is produced in the heart, kidneys, and lungs. ProS is mainly produced in the liver11. TAM recognizes of apoptotic cells in such a way that the N-terminal of Gas6/ProS binds to the PtdSer on an apoptotic cell and the C-terminal of Gas6/ProS binds to TAM receptors that anchored on the surface of phagocytes. Together with the other receptors, engulfment of apoptotic cells occurs12. Though Mer can bind to both the ligands ProS and Gas6, we found that Gas6 appears to be the sole ligand for Mer-mediated macrophage phagocytosis of apoptotic cells, which can be blocked by anti-Mer antibody13. Macrophages are professional phagocytes. Rapid clearance of apoptotic cells by macrophages is important for the inhibition of inflammation and autoimmune responses against intracellular antigens. Mer receptor tyrosine kinase is critical for the macrophage engulfment and efficient clearance of apoptotic cells14. In mouse spleen, Mer mainly expresses on the marginal zone and tangible body macrophages13.
The protocol presented here describes a basic method to induce cell apoptosis and demonstrate ways to measure the process and the efficiency of efferocytosis. These protocols can be readily adapted to study efferocytosis by other cell types in engulfment of apoptotic cells of different origins.

<table border="0" cellpadding="0" cellspacing="0" style="width:832px;" width="832">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Ack lysing buffer</td>
			<td style="width:131px;">GIBCO</td>
			<td style="width:135px;">A10492</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Annexin V/7-AAD</td>
			<td style="width:131px;">BD Pharmingen</td>
			<td style="width:135px;">559763</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Anti-Mer antibody</td>
			<td style="width:131px;">R&#38;D Systems</td>
			<td style="width:135px;">BAF591</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">CD11b-PE (clone M1/70)</td>
			<td style="width:131px;">BD Pharmingen</td>
			<td style="width:135px;">553311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">CFSE</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:135px;">C1157</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">DMSO</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:135px;">D-2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">EDTA (0.5 mM)</td>
			<td style="width:131px;">GIBCO</td>
			<td style="width:135px;">15575-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">FACS tubes</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:135px;">352017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Frosted slides</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:135px;">12-552-343</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Horse Serum (Heat-inactivated)</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:135px;">26050088</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Lidocaine</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:135px;">L-5647</td>
			<td style="width:275px;">Prepare 1% buffer in 1x PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">PBS, 1x</td>
			<td style="width:131px;">Corning</td>
			<td style="width:135px;">21040CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">RPMI-1640</td>
			<td style="width:131px;">Corning</td>
			<td style="width:135px;">10040CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">RXDX-106</td>
			<td style="width:131px;">Selleck Chemicals</td>
			<td style="width:135px;">CEP-40783</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:292px;">Staurosprine (100mg)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:135px;">BP2541-100</td>
			<td style="width:275px;">Add 214.3 ml of DMSO into 100mg to make 1mM stocking solution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:292px;">Thioglycolate Medium Brewer Modified</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:135px;">243010</td>
			<td style="width:275px;">Prepare 3% thioglycolate buffer in 1&#180;PBS, autoclaved, and store in the dark for 3 months.</td>
		</tr>
	</tbody>
</table>

experimental analysis of apoptotic thymocyte engulfment by macrophages

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Analysis of peritoneal macrophage-mediated engulfment of apoptotic thymocytes. Peritoneal macrophages and apoptotic cells were prepared and co-cultured as described in the protocol. Macrophages were detached and stained with PE conjugated anti-CD11b antibody for 20 min on ice. Macrophages were then washed and processed in a flow cytometer. As seen, there is no CFSE positive macrophage in the bottom right quadrant when no apoptotic cells were added into the culture (Figure 1<...

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Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...

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JoVE Journal

Immunology and Infection

6.9K Views.  University of Cincinnati. Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

Video: Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...