This work is supported by grant R01HL116826 to X. Zhao.

This protocol follows the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Eastern Virginia Medical School.
1. Fluorescent Beads Phagocytosis
<ol>
	<li>Euthanize the mouse (C57BL/6J, 6 weeks old, female) by CO2 asphyxiation as per IACUC protocols for the ethical euthanasia of animals.</li>
	<li>Lay the mouse belly-up on a dissection board covered with paper towels. Pin its paws down with its limbs spread-eagle and hook a string under its front teeth to pull its head back so that the trachea is positioned straight and level.</li>
	<li>Wet the mouse&#39;s throat, chest, and belly with 70% ethanol to disinfect and prevent the fur from sticking to the tools.</li>
	<li>Using regular forceps, pull up the skin at the centerline of the body, and cut with surgical scissors up the centerline to the top of the throat.</li>
	<li>Using the blunt end of standard surgical scissors, carefully move away the muscle and connective tissues on the throat and use spring scissors (microscissors) to expose the trachea.</li>
	<li>Gripping a cartilage ring with the forceps, carefully make a small incision (~1.5 mm), using microscissors, on the ventricle face of the trachea and insert an 18 G cannula&#160;into the trachea.</li>
	<li>Gently lavage 3 mL of phosphate-buffered saline (PBS), 1 mL at a time. Each time gently withdraw the fluid into the syringe and reinfuse it back into the lung, 3x in succession. After collecting the BALF, perform cervical dislocation to ensure euthanasia.&#160;Transfer the collected PBS (~2.8 mL), which is bronchoalveolar lavage fluid (BALF), to a tube, centrifuge at 1,000 x g for 10 min, and collect the pellet. Add 1 mL of fresh PBS to the tube and centrifuge at 1,000 x g for 10 min to wash the debris and collect the pelleted alveolar macrophages.</li>
	<li>Resuspend the pellet in 2 mL of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with 10% nonheat-inactivated fetal bovine serum (FBS) and culture primary alveolar macrophages in the same media for 2 days on a glass-bottom dish at 37 &#176;C in a humidified atmosphere.</li>
	<li>Aspirate the old media, wash it with 1 mL of PBS, and add 2 mL of fresh media. Add FITC beads (carboxylated latex beads, 2 &#181;m in diameter, 50 beads/cell) and incubate for 1 h at 37 &#176;C in a humidified atmosphere.</li>
	<li>Wash extensively with PBS (1 mL at a time, for a total of five washes) to remove extracellular beads. Image 100 cells randomly and count the cells with intracellular beads (488 nm). 
	&#8203;NOTE: Phagocytic indexes are the number of ingested beads divided by the total number of macrophages; the percentage of phagocytic cells is the number of macrophages that ingest at least one bead divided by the total number of macrophages16.
	<ol>
		<li>Alternatively, after a 1 h incubation with beads, wash the cells with 3 mL of PBS and process them for flow cytometry for the quantification of phagocytosis. Similarly, process AMs without beads as unstained cells or control cells. Calculate the percentage positivity and mean fluorescence intensity, using flow cytometry software, by selecting those options in the software.</li>
	</ol>
	</li>
</ol>
2. Fc&#947;R- and CR-mediated Phagocytosis
<ol>
	<li>For the opsonization, incubate 2 x 108 sheep red blood cells (SRBCs) with 50 &#181;L of rabbit anti-SRBC-IgM or 50 &#181;L of rabbit anti-SRBC-IgG for 30 min at room temperature11.</li>
	<li>Incubate IgM-opsonized SRBCs with 50 &#181;L of C5-deficient (C5D) human serum for 30 min at 37 &#176;C to fix the complement fragments C3b and C3bi on IgM-coated SRBCs.</li>
	<li>Seed murine macrophage cells (MH-S cells) (10,000 cells/well) in a 96-well plate and incubate overnight to get a ~70% confluence. Add 100 &#181;L of 1 x 107/mL opsonized SRBCs to each well of MH-S cells and incubate for 1 h at 37 &#176;C. Wash unbound SRBCs very quickly (~1 min) with 100 &#181;L of ammonium chloride-potassium (ACK) lysis buffer.</li>
	<li>Lyse the cells with 0.1% SDS and add 50 &#181;L of 2,7-diaminofluorene (DAF) containing 3% hydrogen peroxide and 6 M urea. Measure the absorbance of the hemoglobin-catalyzed fluorene blue formation at 620 nm.</li>
	<li>Determine the number of SRBCs by using a standard curve at 620 nm absorbance values with a known number of SRBCs. Similarly, process MH-S cells incubated with nonopsonized SRBCs to use as negative controls.</li>
</ol>
3. PRR-mediated Phagocytosis
<ol>
	<li>Follow steps 1.1-1.9 for the isolation and culturing of mouse primary alveolar macrophages.</li>
	<li>After 2 days, remove the media, wash the cells with 1 mL of PBS, and add 500 &#181;L of fresh media containing Alexa Fluor-488-conjugated zymosan-A bioparticles (100 particles/dish).</li>
	<li>Incubate for 1 h at 37 &#176;C. Stop the phagocytosis by adding 500 &#181;L of ice-cold PBS.</li>
	<li>Wash the cells extensively with PBS (1 mL at a time, for a total of five washes). Fix the cells with 4% paraformaldehyde for 10 min at room temperature.</li>
	<li>Wash the cells extensively with PBS (1 mL at a time, for a total of five washes) and keep the cells in 500 &#181;L of PBS.</li>
	<li>Image the cells under differential interference contrast and a fluorescent channel at 488 nm. Count AMs containing zymosan-A bioparticles and determine the percentage of phagocytosis.</li>
</ol>
4. In Vivo Phagocytosis by Alveolar Macrophages
NOTE: Inoculate P. aeruginosa GFP on a nutrient agar plate and incubate the plate at 37&#176;C overnight. On the next day, inoculate the single colony to 2 mL of nutrient broth and grow the bacteria at 37&#176;C overnight.
<ol>
	<li>The next day, anesthetize mice with an intraperitoneal administration of 100 mg/kg ketamine and 10 mg/kg xylazine. Confirm proper anesthetization via a lack of response to the toe pinch.</li>
	<li>Lay the mouse on a flat board with a rubber band across the upper incisors and place it in a semirecumbent (45&#176;) position with the ventral surface and rostrum facing upward. Using curved forceps, partially retract the tongue. Using a microsprayer, intratracheally administer 50 &#181;L (5 x 106 colony-forming units [CFU])17 of P. aeruginosa GFP into the lungs of the anesthetized mice.</li>
	<li>After 1 h of infection, follow steps 1.1-1.7.</li>
	<li>Resuspend the cells in PBS and cytocentrifuge them (1,000 x g, 1 min at room temperature) onto a glass slide.</li>
	<li>Differentially stain the cytospin slides for alveolar macrophages, neutrophils, and lymphocytes, according to the manufacturer&#39;s instructions.</li>
	<li>Randomly select 100 AMs, count the AMs containing intracellular bacteria, and determine the percentage of phagocytosis.</li>
</ol>
5. In Vivo Bacteria Clearance Using P. aeruginosa
<ol>
	<li>Inoculate P. aeruginosa on a P. aeruginosa isolation agar plate and incubate the plate at 37&#176;C overnight. Inoculate the single colony to 2 mL of lysogeny broth (LB) and grow the bacteria at 37&#176;C overnight. Calculate the CFU, using the following formula. 
	 
	CFU/mL = (number of colonies x dilution factor)/volume of the culture plate. 
	 
	Dilute the culture with PBS to get the desired CFU/mL.</li>
	<li>In trial 1, intratracheally inject a sublethal dose of ~2.5 x 105 CFU/mL P. aeruginosa into anesthetized wild-type (WT) and TRIM72KO mice, as stated in step 4.2. Measure the body weight daily for 6 days.</li>
	<li>In trial 2, inject a second dose of P. aeruginosa (5 x 105 CFU/mL) into the mice that survived in trial 1 and measure the body weight for 4 days.</li>
	<li>In trial 3, using a different set of mice, inject WT and TRIM72KO mice with a lethal dose (3 x 107 CFU/mL) of P. aeruginosa and record the mortality within 2 days of injection.&#160;Animals showing signs of severe stress such as significant body weight loss, hunched back, anorexia, or dyspnea will be assessed by the attending vets. Untreatable animals will be sacrificed immediately.</li>
	<li>Either at death or after euthanasia at day 2 after the injection, collect the whole-lung tissue for the quantification of the lung bacterial burden at peak infection.</li>
	<li>To test the lung bacterial burden, add 200 &#181;L of normal saline to the lung tissues and homogenize them, using a previously tested setting on an electronic homogenizer that completely disrupts the lung tissue without breaking bacteria. Adjust the total volume of the lung homogenate to 1 mL and plate 100 &#181;L of lung lysate on Pseudomonas isolation agar plates at 10-fold serial dilutions.</li>
	<li>Incubate the plates at 37 &#176;C for 24 h and count the bacterial colonies to determine the CFU per whole lung.</li>
</ol>
6. Statistical Analysis
<ol>
	<li>Use Student&#39;s t-test to determine the statistical significance of the difference between the two groups. Consider a difference statistically significant when p &#60; 0.05. All data are presented as means &#177; standard error of the mean (SEM).</li>
</ol>

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The authors have nothing to disclose.

While performing a gas exchange function, the lung persistently confronts foreign particles, pathogens, and allergens. AMs provide the first line of defense by virtue of their main function, namely phagocytosis. AMs also coordinate with other immune cells in destroying the pathogens and in the resolution of inflammation. Here, we described methods for specifically assessing phagocytosis by AMs isolated from the mouse lung. The protocol presented in this manuscript explains a detailed study of phagocytosis both in vivo an...

AMs are the major resident phagocytes in the alveoli at the resting stage and one of the major players of innate immune responses through the recognition and internalization of inhaled pathogens and foreign particles1,2. It has been reported that AMs are essential for the rapid clearance of many pulmonary pathogens such as P. aeruginosa and Klebsiella pneumonia3,4, so a deficiency in AM phagocytosis often results in respiratory infections, such as acute pneumonia, which cause higher mortality and morbidity rates.
AMs also initiate innate inflammatory responses in the lung by producing cytokines and chemokines such as TNF-&#945; and IL-1&#946;, which crosstalk with other cells of the alveolar environment to produce chemokines and recruit inflammatory neutrophils, monocytes, and adaptive immune cells in the lung5. For example, IL-1&#946; produced by AMs helps to prime the release of the neutrophil chemokine CXCL8 from epithelial cells6. Moreover, AMs contribute to the phagocytosis of apoptotic polymorphonuclear leukocytes (PMNs), failure of which leads to the sustained leakage of intracellular enzymes from PMNs to the surrounding tissue, resulting in tissue damage and prolonged inflammation7,8,9.
Phagocytosis by the AMs is mediated by a direct recognition of pathogen-associated molecular patterns at the pathogen surface by the pattern recognition receptors (PRRs) of the AMs or by the binding of opsonized pathogens with immune effector receptors of the AMs10. For the latter, AMs can recognize the targets opsonized with immunoglobulin (IgG) through their Fc&#947; receptors (Fc&#947;R) or the pathogens coated with complement fragments, C3b and C3bi, through their complement receptors (CR)11. Among complement receptors, the CR of the immunoglobulin superfamily (CRIg) is selectively expressed in tissue macrophages12, and a recent finding highlighted the role of the CRIg in AM phagocytosis in the context of P. aeruginosa pneumonia13.
Many original studies use methods to evaluate macrophage phagocytosis to describe the molecular mechanisms of macrophage function14,15. However, methods like in vivo phagocytosis require a precise quantification of phagocytosis. Here, we summarize a detailed methodology for both in vitro and in vivo phagocytosis using fluorescein isothiocyanate (FITC)-glass beads and P. aeruginosa green fluorescent protein (GFP), respectively. Further, we explain the method of differentiating among PRR-, CR-, and Fc&#947;R-mediated phagocytosis. Finally, we report a method to characterize bacterial clearance in mouse with respect to P. aeruginosa pneumonia.

<table border="0" cellpadding="0" cellspacing="0" style="width:809px;" width="809">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">18-G Needle</td>
			<td style="width:179px;">Nipro Medical&#160;</td>
			<td style="width:143px;">CI+1832-2C</td>
			<td style="width:272px;">Molecular Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2,7-diaminofluorene (DAF)</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:143px;">D17106</td>
			<td>Molecular Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70% Ethanol</td>
			<td style="width:179px;">Decon Labs Inc.</td>
			<td style="width:143px;">18C27B</td>
			<td>Analytical grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well plate</td>
			<td style="width:179px;">Corning</td>
			<td style="width:143px;">3603</td>
			<td>Cell Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ACK lysis buffer</td>
			<td style="width:179px;">Life Technologies</td>
			<td style="width:143px;">A10492</td>
			<td>Molecular Biology grade</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa fluor-488 Zymosan-A-bioparticle</td>
			<td style="width:179px;">Thermofisher Scientific</td>
			<td style="width:143px;">Z23373</td>
			<td>Molecular Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C5 deficient serum&#160;</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:143px;">C1163</td>
			<td>Biochemical reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:179px;">Labnet International</td>
			<td style="width:143px;">C0160-R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cytospin 4 Cytocentrifuge</td>
			<td style="width:179px;">Thermofisher Scientific</td>
			<td style="width:143px;">A78300101 Issue 11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM Cell Culture Media</td>
			<td style="width:179px;">Gibco</td>
			<td style="width:143px;">11995-065</td>
			<td>Cell Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:179px;">Atlanta Biologicals</td>
			<td style="width:143px;">S11550</td>
			<td>Cell Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Flow Cytometer</td>
			<td style="width:179px;">BD Biosciences</td>
			<td style="width:143px;">FACSCalibur</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Flow Jo Software</td>
			<td style="width:179px;">FlowJo, LLC</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forceps</td>
			<td style="width:179px;">Dumont</td>
			<td style="width:143px;">0508-SS/45-PS-1</td>
			<td>Suitable for laboratory animal dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FITC-carboxylated latex beads</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:143px;">L4530</td>
			<td>Cell Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GFP-P. aeruginosa</td>
			<td style="width:179px;">ATCC</td>
			<td>101045GFP</td>
			<td>Suitable for&#160; cell infection assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass bottom dish</td>
			<td style="width:179px;">MatTek Corp.</td>
			<td>P35G-0.170-14-C</td>
			<td>Cell Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">High-Pressure Syringe</td>
			<td style="width:179px;">Penn-Century</td>
			<td>FMJ-250</td>
			<td>Suitable for laboratory animal use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Homogenizer</td>
			<td style="width:179px;">Omni International</td>
			<td>TH-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrogen peroxide</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:143px;">H1009</td>
			<td>Analytical grade</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Inverted Fluorescence Microscope</td>
			<td style="width:179px;">Olympus</td>
			<td style="width:143px;">IX73</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine Hydrochloride</td>
			<td style="width:179px;">Hospira</td>
			<td style="width:143px;">CA-2904</td>
			<td>Pharmaceutical grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Shandon Kwik-Diff Stains</td>
			<td style="width:179px;">Thermofisher Scientific</td>
			<td style="width:143px;">9990700</td>
			<td>Cell Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB Agar</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:143px;">BP1425</td>
			<td>Molecular Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB Broth</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:143px;">BP1427</td>
			<td>Molecular Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MicroSprayer Aerosolizer</td>
			<td style="width:179px;">Penn-Century</td>
			<td style="width:143px;">IA-1C</td>
			<td>Suitable for laboratory animal use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraformaldehyde</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:143px;">P6148</td>
			<td>Reagent grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:179px;">Gibco</td>
			<td style="width:143px;">20012-027</td>
			<td>Cell Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit anti-SRBC-IgG&#160;</td>
			<td style="width:179px;">MP Biomedicals</td>
			<td style="width:143px;">55806</td>
			<td>Suitable for immuno-assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit anti-SRBC-IgM&#160;</td>
			<td style="width:179px;">Cedarline Laboratories</td>
			<td style="width:143px;">CL9000-M</td>
			<td>Suitable for immuno-assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scissors</td>
			<td style="width:179px;">Miltex</td>
			<td style="width:143px;">5-2</td>
			<td>Suitable for laboratory animal dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small Animal Laryngoscope</td>
			<td style="width:179px;">Penn-Century</td>
			<td style="width:143px;">LS-2</td>
			<td>Suitable for laboratory animal use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Dodecyl Sulfate (SDS)</td>
			<td style="width:179px;">BioRad</td>
			<td style="width:143px;">1610301</td>
			<td>Analytical grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring Scissors (Med)</td>
			<td style="width:179px;">Fine Science Tools</td>
			<td style="width:143px;">15012-12</td>
			<td>Suitable for laboratory animal dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring Scissors (Small)</td>
			<td style="width:179px;">Fine Science Tools</td>
			<td style="width:143px;">91500-09</td>
			<td>Suitable for laboratory animal dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sheep red blood cells (SRBCs)&#160;</td>
			<td style="width:179px;">MP Biomedicals</td>
			<td style="width:143px;">55876</td>
			<td>Washed, preserved SRBCs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:143px;">U5378</td>
			<td>Molecular Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine&#160;</td>
			<td style="width:179px;">Akorn Animal Health</td>
			<td style="width:143px;">59399-110-20</td>
			<td>Pharmaceutical grade</td>
		</tr>
	</tbody>
</table>

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.

We first performed the experiment to analyze phagocytosis by mouse primary AMs. Throughout all analyses, we compared AMs isolated from WT and TRIM72KO mice. As shown in Figure 1A, fluorescence microscopy revealed that phagocytosis of FITC-glass beads by mouse primary AMs occurs after 1 h of incubation. Figure 1B shows the analysis of phagocytosis by flow cytometry. The quantification o...

Watch this Scientific Journal Video about Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice at JoVE.com

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

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 ...

alveolar-macrophage-phagocytosis-and-bacteria-clearance-in-mice

Research

JoVE Journal

Immunology and Infection

11.0K Views.  Eastern Virginia Medical School. 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.

Video: Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

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 ...

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

We herein describe a phagocytosis assay using the dispersed embryonic cells of Drosophila. It enables us to easily and precisely quantify in vivo phagocytosis levels, and to identify new molecules required for the phagocytosis of apoptotic cells.

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

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.

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.

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

Assaying for Inorganic Polyphosphate in Bacteria

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many bacteria. There are a variety of methods for quantifying polyP in biological materials, many of which are either labor-intensive or insensitive, limiting their usefulness. We present here a streamlined method for polyP quantification in bacteria, using a silica membrane column extraction optimized for rapid processing of multiple samples, digestion of polyP with the polyP-specific exopolyphosphatase ScPPX, and detection of the resulting free phosphate with a sensitive ascorbic acid-based colorimetric assay. This procedure is straightforward, inexpensive, and allows reliable polyP quantification in diverse bacterial species. We present representative polyP quantification from the Gram-negative bacterium (Escherichia coli), the Gram-positive lactic acid bacterium (Lactobacillus reuteri), and the mycobacterial species (Mycobacterium smegmatis). We also include a simple protocol for nickel affinity purification of mg quantities of ScPPX, which is not currently commercially available.

We describe a simple method for rapid quantification of inorganic polyphosphate in diverse bacteria, including Gram-negative, Gram-positive, and mycobacterial species.

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many ...

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing bronchoalveolar lavage (BAL) fluid. The BAL fluid is rich in cells and can thus provide a 'snapshot' of the pulmonary immune milieu. CD4 T cells are the best characterized HIV reservoirs, while there is strong evidence to suggest that tissue macrophages, including alveolar macrophages (AMs), also serve as viral reservoirs. However, much is still unknown about the role of AMs in the context of HIV reservoir establishment and maintenance. Therefore, developing a protocol for processing BAL fluid to obtain cells that may be used in virological and immunological assays to characterize and evaluate the cell populations and subsets within the lung is relevant for understanding the role of the lungs as HIV reservoirs. Herein, we describe such a protocol, employing standard techniques such as simple centrifugation and flow cytometry. The CD4 T cells and AMs may then be used for subsequent applications, including immunophenotyping and HIV DNA and RNA quantification.

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

We describe a method for processing bronchoalveolar lavage fluid and matched peripheral blood from chronically HIV-infected individuals on antiretroviral therapy to assess pulmonary HIV reservoirs. These methods result in the acquisition of highly pure CD4 T cells and alveolar macrophages that may subsequently be used for immunophenotyping and HIV DNA/RNA quantifications by ultrasensitive polymerase chain reaction.

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing ...

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce pulmonary inflammation, but little is known regarding how exposure alters processes important to the resolution of inflammation. Efferocytosis is a resolution process, whereby macrophages phagocytize apoptotic cells. The purpose of this protocol is to measure alveolar macrophage efferocytosis following O3-induced lung injury and inflammation. Several methods have been described for measuring efferocytosis; however, most require ex vivo manipulations. Described in detail here is a protocol to measure in vivo alveolar macrophage efferocytosis 24 h after O3 exposure, which avoids ex vivo manipulation of macrophages and serves as a simple technique that can be used to accurately represent perturbations in this resolution process. The protocol is a technically non-intensive and relatively inexpensive method that involves whole-body O3 inhalation followed by oropharyngeal aspiration of apoptotic cells (i.e., Jurkat T cells) while under general anesthesia. Alveolar macrophage efferocytosis is then measured by light microscopy evaluation of macrophages collected from bronchoalveolar (BAL) lavage. Efferocytosis is finally measured by calculating an efferocytic index. Collectively, the outlined methods quantify efferocytic activity in the lung in vivo while also serving to analyze the negative health effects of O3 or other inhaled insults.

This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis.

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...