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

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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

23.6K Views.  St. Joseph's Hospital and Medical Center. Deze mededeling beschrijft methodologieën voor isolatie en cultuur van de alveolaire macrofagen van mens en lymfkliertest modellen voor experimentele doeleinden.

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

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Bioengineering

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. ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

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 ...

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 ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by electrical impulses generated in sinoatrial node (SAN) and then conducted to ventricles via atrioventricular node (AVN). To further study the role of resident macrophages in cardiac conduction system, a proper isolation of resident macrophages from SAN and AVN is necessary, but it remains challenging. Here, we provide a protocol for the reliable microdissection of the SAN and AVN in murine hearts followed by the isolation and culture of resident macrophages.
Both, SAN which is located at the junction of the crista terminalis with the superior vena cava, and AVN which is located at the apex of the triangle of Koch, are identified and microdissected. Correct location is confirmed by histologic analysis of the tissue performed with Masson's trichrome stain and by anti-HCN4.
Microdissected tissues are then enzymatically digested to obtain single cell suspensions followed by the incubation with a specific panel of antibodies directed against cell-type specific surface markers. This allows to identify, count, or isolate different cell populations by fluorescent activated cell sorting. To differentiate cardiac resident macrophages from other immune cells in the myocardium, especially recruited monocyte-derived macrophages, a delicate devised gating strategy is needed. First, lymphoid lineage cells are detected and excluded from further analysis. Then, myeloid cells are identified with resident macrophages being determined by high expression of both CD45 and CD11b, and low expression of Ly6C. With cell sorting, isolated cardiac macrophages can then be cultivated in vitro over several days for further investigation. We, therefore, describe a protocol to isolate cardiac resident macrophages located within the cardiac conduction system. We discuss pitfalls in microdissecting and digesting SAN and AVN, and provide a gating strategy to reliably identify, count and sort cardiac macrophages by fluorescence-activated cell sorting.

The protocol presented here provides a step-by-step approach for the isolation of cardiac resident macrophages from the sinoatrial node (SAN) and atrioventricular node (AVN) region of mouse hearts.

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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