We thank Clare Prendergast for assistance with editing the manuscript. DKN is supported by a research grant (#2095) from the Flinn Foundation and TM is supported by grants from National Institutes of Health (R01HL056643 and R01HL092514). DKN developed the methods, designed the study and wrote the manuscript; OM assisted with animal studies and clinical sample procurement; SB assisted with Flow cytometric analysis and cell sorting; TM supervised the studies and reviewed the manuscript.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Review Board (IRB) at St Joseph&#39;s Hospital and Medical Center.
1. Isolation of AMs from Murine Bronchoalveolar Lavage (BAL) Fluid
<ol>
	<li>Anaesthetize an eight-week-old C57BL/6 mouse with ketamine (87.5 mg/kg body weight) and xylazine (12.5 mg/kg body weight) cocktail via an intraperitoneal injection. Proceed when mouse attains surgical anesthesia with loss of reflexes and relaxation of muscles.</li>
	<li>Place the mouse on a dissection surface with the ventral side facing up. Apply ophthalmic vet ointment on eyes to prevent dehydration and wipe the entire ventral surface of mouse with sterile alcohol prep pads saturated with 70% isopropanol to disinfect.</li>
	<li>Mount the mouse with all four legs restrained.</li>
	<li>Gently open the abdominal cavity with micro dissection tools. Care must be taken to open as much area as possible without damaging any visceral organs or creating overhanging tissue.</li>
	<li>Gently open the thoracic cavity by slowly incising the rib cage through the mediastinum. Ideally, the rib cage can be excised from side to side. Extreme care must be taken not to injure the pleura of lungs while opening the thoracic cavity.</li>
	<li>Locate the inferior vena cava and make an incision to bleed the mouse. Use absorbent cotton pads to soak up blood.</li>
	<li>Inject 10 mL ice-cold phosphate buffered saline (PBS) into the right ventricle (using 10 mL syringe and 25G needle) to flush circulating blood cells. Absorb the flow of blood with absorbent cotton pads. Once completely perfused, the lungs will appear blanched and are then ready for lavage.</li>
	<li>Gently cut open the skin and muscle in the neck to expose the airway. Carefully excise the adjoining muscles, cartilages, and fat tissues without damaging the trachea.</li>
	<li>Make a small incision (&#60;2 mm) on the trachea posterior to the larynx. This incision should be just enough to insert a catheter into while the trachea is still attached to the larynx. Insert a 1-inch 22G catheter without a needle into the trachea towards the lungs. Secure the catheter with a silk braided suture (4-0; non-absorbable) with a square knot. 
	NOTE: The catheter needs to be trimmed based on the mouse size. Extremely short or long catheters tend to leak and/or tear the airway during lavage. Catheter length should be such that when fully inserted the tip remains well within the trachea and not reach the primary bronchi.</li>
	<li>Attach a 1-mL syringe filled with ice-cold BAL buffer (Ca2+ and Mg2+ free PBS + 1 mM Ethylenediaminetetraacetic acid, EDTA) to the catheter and slowly instill the buffer into the lungs. This will inflate the lungs. Keep the syringe attached to the catheter for five seconds and then aspirate the lavage fluid by gently pulling the piston. This will deflate the lungs. Collect the fluid in a 15 mL conical tube placed on ice.</li>
	<li>Repeat Step 1.10 for nine more times and pool the lavage fluid. Do not exceed 1 mL buffer per flush, as that may exceed the lung capacity and lead to its rupture.</li>
	<li>Euthanize the mouse with CO2 overdose by IACUC approved protocol.</li>
	<li>Centrifuge the 10 mL BAL fluid at 250 x g at 4 &#176;C for 10 minutes. The cell pellet contains BAL cells. 
	Caution: The pellet may be very small, so exercise caution when aspirating the supernatant.</li>
	<li>Resuspend the BAL cells in 100 &#956;L of flow/sorting buffer (PBS + 2% Heat inactivated fetal bovine serum (FBS) + 1 mM EDTA + 25 mM N-2-Hydroxyethylpiperazine-N&#39;-2-ethanesulphonic acid (HEPES)).</li>
	<li>Block the nonspecific antibody binding to Fc receptor by adding mouse Fc block (clone 2.4G2, 10 &#956;g/mL) and incubating for 20 minutes on ice.</li>
	<li>Perform antibody staining along with fluorescence minus one (FMO) and single-stain controls11,25. Evaluate the BAL cells by a cell analyzer (see Table of Materials) followed by single-cell flow cytometry analysis by appropriate analysis software (see Table of Materials).</li>
	<li>Isolate AMs by a fluorescence-activated cell sorter (see Table of Materials) directly into 1 mL mouse AM culture medium when AMs are intended for in vitro culture or in vivo cell transfer. Alternatively, sort AMs into 1 mL Lysis buffer (see Table of Materials) supplemented with 10 &#956;L/mL &#946;-Mercaptoethanol for RNA extraction.</li>
</ol>
2. Isolation of AMs from Mouse Lung, Single-cell Suspension
<ol>
	<li>Anaesthetize an eight-week-old C57BL/6 mouse with ketamine (87.5 mg/kg body weight) and xylazine (12.5 mg/kg body weight) cocktail via an intraperitoneal injection. Proceed when mouse attains surgical anesthesia with loss of reflexes and relaxation of muscles.</li>
	<li>Place the mouse on a dissection surface with the ventral side facing up. Apply ophthalmic vet ointment on eyes to prevent dehydration and wipe the entire ventral surface of mouse with sterile alcohol pads saturated with 70% isopropanol to disinfect.</li>
	<li>Mount the mouse with all four legs restrained.</li>
	<li>Gently open the abdominal cavity with micro dissection tools. Care must be taken to open as much area as possible without damaging any visceral organs or creating overhanging tissue.</li>
	<li>Gently open the thoracic cavity by slowly incising the rib cage through the mediastinum. Ideally, the rib cage can be excised from side to side. Extreme care must be taken not to injure the pleura of lungs while opening the thoracic cavity.</li>
	<li>Locate the inferior vena cava and make an incision to bleed the mouse. Use absorbent cotton pads to soak up blood.</li>
	<li>Inject 10 mL ice-cold PBS into the right ventricle (using 10 mL syringe and 25G needle) to flush circulating blood cells. Once completely perfused, the lungs will appear blanched and are ready for harvest.</li>
	<li>Dissect out the lungs by severing the trachea, blood vessels and ligaments into a 15 mL conical tube with 5 mL of cold Dulbecco&#39;s Modified Eagle&#39;s medium (DMEM). Maintain it on ice until the next step.</li>
	<li>Euthanize the mouse with CO2 overdose by IACUC approved protocol.</li>
	<li>Transfer the perfused lungs into a sterile and pyrogen-free 60 mm culture dish with 3 mL DMEM. With help of dissecting forceps tease out the airways and other hard non-lung tissue material, if present. Mince the lung tissue with a scalpel to &#60;1 mm size.</li>
	<li>Add 300 &#181;g/mL Liberase TL and 5 U/mL DNase I, mix by pipetting and incubate at 37&#176; C in an incubator for 25 minutes. Gently mix once by pipetting after 10 minutes.</li>
	<li>Pass the dissociated lungs through a 100 &#181;m cell strainer installed on a 50 mL conical tube. Use the back of a plunger from 1 mL syringe to mash up cell clumps on the filter. Wash the filter with 20 mL wash buffer (PBS + 2% Heat inactivated FBS + 2 mM EDTA).</li>
	<li>Centrifuge at 500 x g at 4 &#176;C for 5 minutes. Discard the supernatant and resuspend the cell pellet in 20 mL wash buffer.</li>
	<li>Repeat steps 2.12 and 2.13 twice, changing the filter and tube each time. Pre-soaking the filter in wash buffer for more than five minutes increases the AM yield. Prepare single-cell suspension by adding 100 &#956;L flow/sorting buffer to the cell pellet.</li>
	<li>Block the nonspecific antibody binding to the Fc receptor by adding mouse Fc block (clone 2.4G2, 10 &#956;g/mL) and incubating for 20 minutes on ice.</li>
	<li>Perform antibody staining along with FMO and single-stain controls11,25. Evaluate the lung cells by a cell analyzer (see Table of Materials) followed by single-cell flow cytometry analysis by analysis software.</li>
	<li>Sort AMs by a cell sorter (see Table of Materials) directly into 1 mL mouse AM culture medium when AMs are intended for in vitro culture or in vivo cell transfer. Alternatively, sort AMs into 1 mL Lysis buffer supplemented with 10 &#956;L/mL &#946;-Mercaptoethanol for RNA extraction.</li>
</ol>
3. Isolation of AMs from Human BAL Fluid
<ol>
	<li>Arrange transfer of the human BAL fluid from the clinic to the laboratory at 4 &#176;C.</li>
	<li>Centrifuge the BAL fluid (10 - 50 mL) at 250 x g at 4 &#176;C for 10 minutes. The cell pellet contains BAL cells11.</li>
	<li>Wash the pellet with 20 mL wash buffer (PBS + 2% Heat inactivated FBS + 2 mM EDTA) and centrifuge at 250 x g at 4 &#176;C for 10 minutes.</li>
	<li>Resuspend the BAL cells are in 100 &#956;L of flow/sorting buffer (PBS + 2% Heat inactivated FBS + 1 mM EDTA + 25 mM HEPES).</li>
	<li>Block the nonspecific antibody binding to the Fc receptor by adding human Fc block (clone Fc1.3070, 25 &#956;g/mL) and incubating for 20 minutes on ice.</li>
	<li>Perform antibody staining along with FMO and single-stain controls11,24. Evaluate the BAL cells by a cell analyzer (see Table of Materials) followed by single-cell flow cytometry analysis by analysis software.</li>
	<li>Sort AMs by a cell sorter (see Table of Materials) directly into 1 mL human AM culture medium or 1 mL Lysis buffer supplemented with 10 &#956;L/mL &#946;-Mercaptoethanol.</li>
</ol>
4. In vitro Culture of AMs
<ol>
	<li>Following the cell sorting, harvest the cells by centrifuging at 250 x g at 4 &#176;C for 10 min.</li>
	<li>Resuspend human AMs in 1 mL human AM culture medium [Roswell Park Memorial Institute medium (RPMI) 1640 supplemented with 10% FBS, 20% L-929 culture supernatant (as a source of macrophage colony stimulating factor (M-CSF), 100 U/mL final concentration), 1 mM sodium pyruvate, 10 mM N-2-hydroxyethylpiperazine-N&#39;-2-ethanesulphonic acid (HEPES), and 1x penicillin/streptomycin (optional)]. Similarly, resuspend mouse AMs in 1 mL mouse AM culture medium [DMEM supplemented with 10% FBS, 20% L-929 culture supernatant (as a source of M-CSF, 100 U/mL final concentration), 1 mM Sodium Pyruvate, 10 mM HEPES, and 1x penicillin/streptomycin (optional)].</li>
	<li>Enumerate the trypan blue-excluding viable cells by a cell counter and adjust the viable cell concentration at 1 x 106/mL. 
	NOTE: Ideally, up to 5 x 105 AMs can be isolated from BAL fluid collected from an 8 - 10 week old C57BL/6 male mouse. The AM yield from the mouse lung digest is variable and may be slightly less than that of the mouse BAL fluid. AM yield from human BAL fluid depends on the volume of the BAL fluid, the total volume of saline used in the lavage, and patient&#39;s clinical condition.</li>
	<li>Based on the yield and experimental necessity, seed the cells in chamber slides, culture dishes or culture flasks. Ideally, seed the AMs at 1 x 105/mL with 10 mL medium in a T25 tissue culture flask.</li>
	<li>Incubate the cells in a 37 &#176;C humidified incubator with 5% CO2 atmosphere. 
	NOTE: AMs will grow as adherent cells and are extremely slow-growing (doubling time &#62;7 days).</li>
	<li>At 24 hours post-seeding, AMs should be adherent and will not be detached by a gentle change of culture medium. Remove the medium by aspiration from one end and replenish with fresh medium pre-warmed to 37 &#176;C. 
	NOTE: The cells can now be used to assess AM physiology or the effects of stimuli. AMs grown in chamber slides can be conveniently stained with appropriate antibodies and are ideal for microscopic visualization. For flow cytometric evaluation, AMs are harvested by incubation with non-enzymatic cell dissociating solution.</li>
	<li>Aspirate the culture medium and add dissociating solution enough to cover the culture surface (approximately 2 mL per T25 flask) and incubate at 37 &#176;C for 5 - 10 minutes. No scraping will be necessary, and it is not recommended to scrape the cells mechanically. Gently tap the sides, add 1 - 2 mL flow/sorting buffer and gently pipette up and down to detach the majority of adherent cells.</li>
	<li>For RNA studies, lyse the cells directly on the culture dish by adding Lysis buffer.</li>
</ol>

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The authors have no conflicts of interest to declare.

AMs are long-living lung-resident macrophages that populate the lungs beginning at birth and enduring over the entire life span26. Their roles in pulmonary physiology7 and pathology12 and their potential to predict of pulmonary autoimmunity24 have been recognized. Because AMs have a long-term presence in lungs11,27 and because they are involved in activation and progre...

The lung microenvironment is a uniquely complex ecosystem with an elaborate air conduit and vasculature. The inhaled air travels through the trachea and through numerous branches of bronchi and bronchioles before reaching the alveoli, where the blood-air gas exchange occurs. Due to direct interaction with the atmosphere, the respiratory surface requires protection from the potentially harmful effects of airborne particles and pollutants. A number of physical, chemical, and immunologic barriers protect the lungs. Notably, deployment of phagocytes at the respiratory surface serves an important first-line defense system. Alveolar macrophages (AMs) are one type of lung-resident phagocytes, and they make up the vast majority of the lung macrophage pool. As their name suggests, AMs are primarily localized to the alveolar lumen and occur as sessile cells that constantly sample the ambient atmosphere and communicate with the alveolar epithelium1. In steady state lungs, more than 95% of the phagocytes in the alveolar space are AMs2, whose composition may alter due to inflammation, infection, or chronic exposure to pollutants.
AMs participate in a wide range of functions that may be local to the lungs and/or of systemic importance. For example, AMs are essential in the development and the optimal functioning of lungs; immune surveillance; and clearance of cellular debris, invading pathogens, and inhaled particles3,4,5,6,7. Targeted depletion of AMs is known to impair clearance of respiratory viruses and bacteria4,8. Besides their role as phagocytes and a first-line defenders of pulmonary homeostasis, AMs are known to function as antigen-presenting cells in eliciting T cell immunity9, potentiating the efficacy of intranasal vaccine10 and influencing lung-restricted autoimmunity after lung transplantation11,12. Deficiency in AM function has been linked to pulmonary alveolar proteinosis (PAP), a condition resulting from a genetic mutation, malignancy or infection that impairs clearance of pulmonary surfactants13,14. Transplantation of AMs is now being explored as a therapeutic approach for treatment of PAP 15,16.
AMs are known to originate during embryogenesis and to persist in the lungs throughout life without being replaced by circulating leukocytes2,17. Although, AM turnover is undetectable in homeostatic lungs, varying levels of AM turnover have been reported in certain clinical conditions including infection by the influenza virus4, myeloablative irradiation18, exposure to endotoxin19, and old age20. AMs are believed to self-renew via a low-grade proliferation17,21, but some recent studies claim that monocytes can give rise to a population of intravascular lung macrophages22,23 under experimental conditions, but functionality of these newly converted pulmonary macrophages have yet to be defined in lung diseases. Furthermore, understanding the threshold of stimulus in the context of AM activation is a potentially interesting area, as the lung attempts to preserve an equilibrium between the inflammatory signals and the immunoregulatory machinery.
The physiologic or pathologic alterations that lead to loss of immune regulation are important to evaluate in various clinical settings (e.g., respiratory infections, inflammatory lung disease and fibrotic lung disease). Nevertheless, AMs are increasingly recognized as indicators or even determinants of pulmonary health11,24. Currently, there are no unified protocols available for harvesting, characterizing, and/or maintaining AMs from humans and preclinical murine models. Lack of a consensus on AM precursors and phenotypes, and absence of a detailed methodology had been the major roadblock in deciphering role(s) of AM in pulmonary health and disease. The following protocol offers a definitive identification, isolation, and in vitro culture strategy that will greatly advance the understanding of AM behavior and facilitate AM-targeted diagnostic and therapeutic studies.

<table border="0" cellpadding="0" cellspacing="0" width="910">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Non-enzymatic cell dissociating solution</td>
			<td style="width:223px;">Millipore-Sigma</td>
			<td style="width:220px;">C5789</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puralube Vet Ointment</td>
			<td style="width:223px;">Dechra</td>
			<td>620300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">22G Catheter&#160;</td>
			<td style="width:223px;">Terumo Medical Products</td>
			<td>SR-OX2225CA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-0 Non-absorbable silk braided suture&#160;</td>
			<td style="width:223px;">Kent Scientific</td>
			<td>SUT-15-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s phosphate buffered saline&#160;</td>
			<td style="width:223px;">Corning</td>
			<td style="width:220px;">21-031-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Fc block&#160;</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysis buffer (PureLink RNA Kit)</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>12183018A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">b-Mercaptoethanol&#160;</td>
			<td style="width:223px;">Millipore-Sigma</td>
			<td>M6250&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACSAria II cell sorter&#160;</td>
			<td style="width:223px;">BD Biosciences</td>
			<td style="width:220px;">644832</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine&#160; (Ketathesia)</td>
			<td style="width:223px;">Henry Schein</td>
			<td style="width:220px;">56344</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine&#160; (AnaSed)</td>
			<td style="width:223px;">Akorn</td>
			<td style="width:220px;">139-236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td style="width:223px;">Corning</td>
			<td style="width:220px;">10-040-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:223px;">Corning</td>
			<td style="width:220px;">10-017-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liberase TL&#160;</td>
			<td style="width:223px;">Millipore-Sigma</td>
			<td style="width:220px;">5401020001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I</td>
			<td style="width:223px;">Millipore-Sigma</td>
			<td>AMPD1-1KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100&#956;m cell strainer&#160;</td>
			<td style="width:223px;">Corning</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Human Fc block</td>
			<td>BD Biosciences</td>
			<td>564220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">EDTA</td>
			<td style="width:223px;">Corning</td>
			<td>46-034-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Countess II Automated Cell Counter</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Trypan Blue Solution</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">HEPES</td>
			<td style="width:223px;">Corning</td>
			<td>25-060-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">L-929 cell line</td>
			<td>American Type Culture Collection</td>
			<td>ATCC,&#160;CCL-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin&#160;</td>
			<td style="width:223px;">Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Pyruvate</td>
			<td style="width:223px;">Corning</td>
			<td>25-000-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T25 Tissue culture flask</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>156367</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 mm culture dish&#160;</td>
			<td style="width:223px;">Millipore-Sigma</td>
			<td>CLS3261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">15 mL Conical tube&#160;</td>
			<td style="width:223px;">Corning</td>
			<td style="width:220px;">352097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">50 mL Conical tube&#160;</td>
			<td style="width:223px;">Corning</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSRFortessa cell analyzer</td>
			<td>BD Biosciences</td>
			<td>657669</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">FlowJo</td>
			<td style="width:223px;">FlowJo</td>
			<td>v10.4</td>
			<td>Analysis Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD45 (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>147709</td>
			<td>Clone I3/2.3, FITC conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD11b (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>101228</td>
			<td>Clone M1/70, PerCP/Cy5.5 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD11c (Mouse)</td>
			<td>BD Biosciences</td>
			<td>565452</td>
			<td>Clone N418, BV 421 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-I-Ab (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>116420</td>
			<td>Clone AF6-120.1, PE/Cy7 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-Siglec-F (Mouse)</td>
			<td style="width:223px;">BD Biosciences</td>
			<td>562757</td>
			<td>Clone E50-2440, PE-CF594 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-Siglec-H (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>129605</td>
			<td>Clone 551, PE conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-F4/80 (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>123118</td>
			<td>Clone BM8, APC/Cy7 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-Ly-6C (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>128035</td>
			<td>Clone HK1.4, BV605 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD64 (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>139311</td>
			<td>Clone X54-5/7.1, BV711 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD24 (Mouse)</td>
			<td style="width:223px;">BD Biosciences</td>
			<td>563115</td>
			<td>Clone M1/69, BV510 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD103 (Mouse)</td>
			<td style="width:223px;">BD Biosciences</td>
			<td>745305</td>
			<td>Clone OX-62, BV650 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD317 (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>127015</td>
			<td>Clone 927, APC conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CXCR1 (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>149029</td>
			<td>Clone SA011F11, BV785 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD45 (Human)</td>
			<td style="width:223px;">Biolegend</td>
			<td>304017</td>
			<td>Clone HI30, AF488 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD11b (Human)</td>
			<td style="width:223px;">Biolegend</td>
			<td>101216</td>
			<td>Clone M1/70, PE/Cy7 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-HLA-DR (Human)</td>
			<td style="width:223px;">Biolegend</td>
			<td>307618</td>
			<td>Clone L243, APC/Cy7 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD169 (Human)</td>
			<td style="width:223px;">Biolegend</td>
			<td>346008</td>
			<td>Clone 7-239, APC conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD206 (Human)</td>
			<td style="width:223px;">Biolegend</td>
			<td>321106</td>
			<td>Clone 15-2, PE conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD163 (Human)</td>
			<td style="width:223px;">Biolegend</td>
			<td>333612</td>
			<td>Clone GHI/61, BV421 conjugated</td>
		</tr>
	</tbody>
</table>

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.

The flow cytometric approach to identify mouse AMs is shown in Figure 1. This includes analysis of a minimum set of surface markers necessary in distinguishing AMs from other lung-resident or lung-infiltrating phagocytes. Differential analysis is required to positively identify AMs from interstitial macrophages, dendritic cells, neutrophils, monocytes, and monocyte-derived pulmonary macrophages that occur in lungs. The following scheme of surface markers can ...

Watch this Scientific Journal Video about Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages at JoVE.com

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.

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 ...

isolation-vitro-culture-murine-human-alveolar

Research

JoVE Journal

Immunology and Infection

23.4K Views.  St. Joseph's Hospital and Medical Center. This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Video: Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

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 ...

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. ...

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 ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Isolation, Transfection, and Culture of Primary Human Monocytes

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4+ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14+ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.

Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. ...