This study is funded by Health Effects Institute Walter A. Rosenblith Award and NIEHS R01ES028829 (to K. M. G). We would like to thank Dr. Dianne Walters (Department of Physiology, ECU) for her assistance with obtaining representative images of alveolar macrophages.

All methods have been approved by the Institutional Animal Care and Use Committee (IACUC) of East Carolina University.
1. Ozone (O3) and filtered air exposures (Day 1)
<ol>
	<li>Place a maximum of 12 female C57BL/6J mice, 8-12 weeks old, in a steel cage (with 12 separate compartments) with wire mesh lids into an O3 exposure chamber.</li>
	<li>Place the thermometer in the exposure chamber with the cage to accurately record the temperature and humidity.</li>
	<li>Turn on the oxygen and ultraviolet (UV) light that is attached to the apparatus. 
	NOTE: Regulated airflow (&#62;30 air changes/h) with controlled temperature (22-23 &#176;C) and relative humidity (45%-50%) is obtained by the O3 apparatus. O3 is generated by the system in the exposure chamber by directing 100% oxygen through a UV light generator, then mixing with a filtered air supply.</li>
	<li>Adjust the O3 concentration to 1 ppm and regularly record O3 levels every 10 min for 3 h. Continuously monitor the temperature and humidity of chamber air, as is the O3 concentration with a UV light photometer. 
	NOTE: Filtered air exposures are performed in a similar apparatus, with only a filtered air supply flowing through the exposure chamber.</li>
	<li>Return the animals to their respective cages with bedding, food, and water ad libitum after 3 h of O3/filtered air exposure.</li>
</ol>
2. Preparation of Jurkat T cell line (Day 2)
NOTE: All procedures should be conducted in a class II biological safety cabinet.
<ol>
	<li>Culture Jurkat T cells in 24 mL of basal cell culture medium + 10% FBS + 5% penicillin/streptomycin at 37 &#176;C + 5% CO2 (Table of Material). Jurkat T cells are a suspension cell line that can be maintained through passaging 1:6-1:8 into pre-warmed culturing media every 3 days. Do not shake.</li>
	<li>To prepare apoptotic cells, grow them to 90% confluency in each flask (which takes 3-4 days to achieve after passaging). For this study, use cells from five T75 flasks to obtain the sufficient number of cells used in this protocol. 
	NOTE: A confluent flask contains about 20-24 million cells.</li>
	<li>Pipette up cells (which is the entire flask) from each flask (approximately 24 mL) and transfer cells to a sterile 50 mL conical tube using a serological pipette. Use multiple conical tubes for multiple flasks.</li>
	<li>Count cells by removing an 11 &#181;L aliquot of cells from the 50 mL conical tube and mix with 11 &#181;L of trypan blue stain and pipette 11 &#181;L onto hemocytometer slides.</li>
	<li>Insert slide into an automated cell counter and record the number of live cells to calculate the total cell count in each flask by multiplying the number of live cells by 24, as each flask contains 24 mL of media.</li>
	<li>Centrifuge the cell suspension at 271&#160;x g for 5 min at room temperature (RT) to pellet cells.</li>
	<li>Discard the supernatant by aspiration and resuspend the cell pellet in media to obtain 3.0 x 106 cells per mL.</li>
	<li>Aliquot 5 mL of cells in 100 mm x 20 mm tissue culture dishes (approximately nine dishes will be used; the total amount of cells in each dish should be ~15 x 106).</li>
	<li>Use one dish for control/unexposed, and the remaining dishes will be exposed to UV.</li>
	<li>Set the UV crosslinker to the correct energy level, press the energy button, and enter &#34;600&#34; using the number pad, which the machine will read as 600 &#181;J/cm2 x 100. 
	NOTE: UV crosslinker energy units is in &#181;J/cm2 x 100; therefore, to achieve 60 millijoules/cm2, convert units to match the UV crosslinker.</li>
	<li>Irradiate all dishes with cells, not including the control, at 60 millijoules (mJ)/cm2 using the UV crosslinker. Remove the top cover of the tissue culture dishes during UV exposure, as UV light will not penetrate the plastic cover.</li>
	<li>Incubate all the dishes in a cell culture incubator, including unexposed control, at 37 &#176;C at 5% CO2&#160;for 4 h.</li>
	<li>Confirm apoptosis by flow cytometry using an apoptosis assay detection kit containing annexin V and propidium iodide (PI) (markers for apoptosis and necrosis, respectively) after 4 h of incubation, per the manufacturer&#39;s instructions26,27. 
	NOTE: Irradiating Jurkat T cells in the UV crosslinker at an energy level of 600 &#181;J/cm2, following a 4 h incubation will lead to &#8805;75% of apoptotic (both early and late) cells having more early apoptotic phenotype than the late apoptotic phenotype. This makes it easier for alveolar macrophages to recognize them and engulf as their membranes are uncompromised, unlike late apoptotic cells, leading to a higher efferocytic index and more accurate imaging of alveolar macrophage efferocytosis in this study.
	<ol>
		<li>Pool 333 &#181;L (1 x 106 cells) of Jurkat T cells from several dishes (both &#34;no UV&#34; and &#34;UV-exposed&#34;) together to use for compensation analysis tubes.</li>
		<li>Aliquot 333 &#181;L of Jurkat T cells in an unstained, annexin V single stain, PI single stain, no UV control, and 600 &#181;J/cm2 UV-exposed labeled flow cytometry tubes.</li>
		<li>Centrifuge tubes at 188&#160;x g for 5 min at RT and decant the supernatant.</li>
		<li>Wash cells by resuspending in 500 &#181;L of cold, 1x phosphate-buffered saline (PBS).</li>
		<li>Centrifuge and pellet cells at 188&#160;x g for 5 min at RT. Discard the supernatant after centrifugation.</li>
		<li>Prepare 400 &#181;L of 1x binding buffer per flow tube by diluting 10x binding buffer with distilled water while cells are centrifuging.</li>
		<li>Prepare annexin V and PI incubation reagent (100 &#181;L per sample/tube) per the manufacturer&#39;s instructions.</li>
		<li>Decant the supernatant after centrifugation and gently resuspend all tubes in 400 &#181;L of 1x binding buffer, then add 100 &#181;L of annexin V incubation reagent to each sample tube. Lastly, add 100 &#181;L of annexin V single stain and PI single stain to their respective tubes, but do not add anything beyond the 1x binding buffer to the unstained tube.</li>
		<li>Incubate tubes in the dark for 15 min at RT.</li>
		<li>Centrifuge all cells at 188&#160;x g for 5 min at RT and decant supernatant.</li>
		<li>Resuspend cells in 400 &#181;L of 1x binding buffer, then analyze samples for apoptosis by flow cytometry. Collect at least 10,000 events per tube to allow accurate representation of staining.</li>
	</ol>
	</li>
	<li>Combine all the irradiated cells from dishes into a 50 mL conical tube and pellet cells by centrifugation at 271&#160;x g for 5 min at RT.</li>
	<li>Discard the supernatant from the tube by aspiration and resuspend cells in 24 mL of sterile phosphate buffered saline (PBS) and pellet cells by centrifugation at 271&#160;x g for 5 min at room temperature.</li>
	<li>Discard the supernatant from the tube by aspiration and resuspend cells in the amount of PBS used for dosing mice approved by IACUC. The dose used is between 5-10 x 106 cells/50 &#181;L per mouse; therefore, for 10 mice, resuspend in 500 &#181;L (number of cells in each dose varies depending on how many cells are cultured for irradiation). 
	NOTE: Makeup at least two additional doses to account for any liquid that may stick to the sides of the pipette tip resulting in the loss of cells.</li>
</ol>
3. Murine oropharyngeal instillation of apoptotic cells (Day 2)
<ol>
	<li>Prepare dosing inoculum of apoptotic cells using a P200 pipette prior to anesthetizing mice to expedite the procedure. As per the institutional guidelines, a volume of 50 &#181;L containing approximately 5-10 x 106 cells is utilized for oropharyngeal (o.p.) instillation to ensure best results.</li>
	<li>Anesthetize mice in a clear chamber with 2% isoflurane at a flow rate of 1 L/min or as per the institutional guidelines. Anesthetize one to two mice at a time; the number is determined by the comfort level of the experimenter. Observe the breathing pattern and confirm deep breaths are visible with 2&#8211;3 s counts between breaths. Check for the depth of anesthesia by the lack of response to the toe pinch.</li>
	<li>Position the mouse in a semi-recumbent supine position. Use a surgical string tied between pegs on a slanted acrylic sheet board to suspend by the maxillary incisors.</li>
	<li>Using a pair of blunt non-ridged forceps, lightly grab and pull the mouse tongue. Instill the apoptotic cells into the oral cavity with a P200 pipette. Dosing is successful when the mice make a crackling noise 1&#8211;2 s after giving the dose. 
	NOTE: Take care to avoid inducing trauma either to the tongue or oropharynx before the apoptotic cell instillation.</li>
	<li>With a gloved finger, gently block the nose until the mouse inhales while the tongue is retracted. Cover the nose until no liquid is visible in the oral cavity and the mouse has taken two or more inhalations. 
	NOTE: As mice are obligate nose breathers, covering the nose helps ensure that the mouse will inhale the apoptotic cells into the lungs.</li>
	<li>Remove the mouse from the inoculation board and return it to the cage to allow recovery from anesthesia. Place the mouse on its back to prevent bedding or debris from blocking the nares during the revovery.</li>
	<li>Wait 90 min after the mouse recovers from anesthesia to allow alveolar macrophages to engulf influx of apoptotic cells after all the mice have awoken from anesthesia. Typically, awakening after anesthesia will take 1&#8211;2 min, which should not affect the outcome/timing of instillation.</li>
</ol>
4. Bronchoalveolar lavage fluid collection and processing (Day 2)
<ol>
	<li>Euthanize each mouse per institutional guidelines 90 min after the mouse has woken up from anesthesia from dosing with apoptotic cells. Here, a lethal injection of ketamine and xylazine is used (90 mg/kg and 10 mg/kg, respectively) followed by excising the diaphragm. 
	NOTE: This time point allows sufficient time for alveolar macrophages to sense and engulf apoptotic cells38.</li>
	<li>Weigh all mice (g) on a scale and record weights. Use the body weight to calculate BAL volume (26.25 mL/kg body weight).</li>
	<li>Place mice on their backs and spray 70% ethanol to sterilize the chest and neck area.</li>
	<li>Make a 2&#8221; longitudinal cut just below the sternum along the entire ventral side with surgical scissors, and while holding the sternum with forceps, nick the diaphragm to allow the lungs to fall back into the chest cavity.</li>
	<li>Cut laterally along the sides of the rib cage to allow the lungs more room to expand when lavaging, then fold the chest cavity back with forceps.</li>
	<li>Make a 1&#34; vertical cut up along vasculature through the neck to expose the trachea.</li>
	<li>Use two forceps to pull muscle and tissue off the trachea and expose it. Avoid additional potential bleeding and cutting the trachea, since it is surrounded by vasculature, longitudinal muscles, and connective tissue.</li>
	<li>Use a needle to make a slit in the trachea (about one-quarter of the distance down from the head) and insert a cannula (18 G x 1.25&#34;) with a syringe pre-loaded with 1x PBS (26.25 mL/kg body weight, ~0.7-1.0 mL in an 8-10 week old female C57Bl/6J mouse) caudally into the trachea.</li>
	<li>Push volume of PBS into the lungs slowly to allow the lungs to inflate then, pull the volume back out into the syringe. Repeat this process a total of 3 times.</li>
	<li>Collect the pooled lavage fluid from each specific mouse in a 15 mL tube.</li>
	<li>Centrifuge the bronchoalveolar lavage at 610&#160;x g for 6 min at 4 &#176;C and collect supernatant into a 1.5 mL tube and freeze at -80 &#176;C. The pellet represents cells from the bronchoalveolar space.</li>
	<li>Remove residual red blood cells in collected BAL fluid by adding 1 mL of ACK RBC lysis buffer to the cell pellet, then vortex well and lyse for 1 min on ice. Afterwards, add 4 mL of PBS to stop the lysis reaction.</li>
	<li>Pellet cells by centrifugation at 610&#160;x g for 6 min at 4 &#176;C and aspirate the supernatant with a vacuum aspirator.</li>
	<li>Resuspend cells in 1 mL of 1x&#160;PBS + 10% FBS to each BAL sample tube. Count cells on a hemocytometer for the quantification of total airspace cells from each sample (no trypan blue). Centrifuge 120 &#181;L of each sample onto slides at 56&#160;x g for 3 min, using medium acceleration and a cytocentrifuge. Dry the slides overnight.</li>
</ol>
5. Calculation of alveolar macrophage efferocytic index (Day 3)
<ol>
	<li>Stain the slides with hematoxylin and eosin to allow for calculation of both efferocytic and differential cell counts, with at least 200 cells counted from each slide.</li>
	<li>View slides under a bright-field setting on a biological microscope (a 20x or 40x objective will work best).</li>
	<li>Calculate the efferocytic index based on the ratio of the number of alveolar macrophages that phagocytosed apoptotic Jurkat T cells to alveolar macrophages without apoptotic cell uptake out of a total 200 macrophages on a cell differential slide. Convert the ratio to a percentage for data input. Use the following equation:</li>
</ol>
<img src="/files/ftp_upload/60109/60109eq1.jpg" />

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+surfactant+protein+a+inhibits+macrophage+reactive+oxygen+intermediate+production+in+response+to+stimuli+by+reducing+NADPH+oxidase+activity.">Pulmonary surfactant protein a inhibits macrophage reactive oxygen intermediate production in response to stimuli by reducing NADPH oxidase activity.</a> Journal of Immunology. 172 (11), 6866-6874 (2004).</li><li>Silveyra, P., Floros, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variant+associations+of+human+SP-A+and+SP-D+with+acute+and+chronic+lung+injury.">Genetic variant associations of human SP-A and SP-D with acute and chronic lung injury.</a> Frontiers in Bioscience. 17, 407-429 (2012).</li><li>Schagat, T. L., Wofford, J. A., Wright, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfactant+protein+A+enhances+alveolar+macrophage+phagocytosis+of+apoptotic+neutrophils.">Surfactant protein A enhances alveolar macrophage phagocytosis of apoptotic neutrophils.</a> Journal of Immunology. 166 (4), 2727-2733 (2001).</li><li>Gomez Perdiguero, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+macrophages+originate+from+yolk-sac-derived+erythro-myeloid+progenitors.">Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors.</a> Nature. 518 (7540), 547-551 (2015).</li><li>Nebbioso, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-resolved+analysis+of+DNA-protein+interactions+in+living+cells+by+UV+laser+pulses.">Time-resolved analysis of DNA-protein interactions in living cells by UV laser pulses.</a> Scientific Reports. 7 (1), 11725 (2017).</li><li>Novak, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+different+UV-emitting+light+sources+in+the+induction+of+T-cell+apoptosis.">Efficacy of different UV-emitting light sources in the induction of T-cell apoptosis.</a> Photochemistry and Photobiology. 79 (5), 434-439 (2004).</li><li>Park, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAI-1+inhibits+neutrophil+efferocytosis.">PAI-1 inhibits neutrophil efferocytosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (33), 11784-11789 (2008).</li><li>Kleeberger, S. R., Reddy, S., Zhang, L. Y., Jedlicka, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+susceptibility+to+ozone-induced+lung+hyperpermeability:+role+of+toll-like+receptor+4.">Genetic susceptibility to ozone-induced lung hyperpermeability: role of toll-like receptor 4.</a> American Journal of Respiratory Cell and Molecular Biology. 22 (5), 620-627 (2000).</li><li>Wesselkamper, S. C., Chen, L. C., Kleeberger, S. R., Gordon, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variability+in+the+development+of+pulmonary+tolerance+to+inhaled+pollutants+in+inbred+mice.">Genetic variability in the development of pulmonary tolerance to inhaled pollutants in inbred mice.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 281 (5), L1200-L1209 (2001).</li></ol>

The authors declare no conflicts of interest.

Efferocytosis is an anti-inflammatory process in which macrophages clear apoptotic cells and debris as well as produce multiple anti-inflammatory mediators9,10,11,12,16,18. Multiple models of efferocytosis have provided insight into how the macrophage is a critical cell in the resolution of inflammation6</su...

The lung is constantly exposed to environmental insults, including air particulates, viruses, bacteria, and oxidant gases that trigger pulmonary inflammation1,2,3. These insults can compromise gas exchange and induce irreversible tissue injury4,5. Alveolar macrophages, which constitute approximately 95% of the immune cells found in murine and human lungs at homeostasis, are critical regulators of pulmonary inflammation after environmental insults1,2,3,4,5. Alveolar macrophages are essential during the host defense by phagocytizing and eliminating pathogens. Recently, alveolar macrophages have been shown to promote tissue homeostasis and the resolution of inflammation through efferocytosis6,7. Efferocytosis is a phagocytic process in which macrophages engulf and eliminate apoptotic cells8,9,10. Efferocytosis also results in the production of mediators (i.e., IL-10, TGF-&#946;, PGE2, and nitric oxide) that further augment the process, resulting in the resolution of inflammation9,10,11,12,16,18. This process is necessary for preventing secondary necrosis and promoting tissue homeostasis12,13,14. Several studies have linked impaired efferocytosis with various chronic lung diseases, including asthma, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis8,9,15,16,17.
O3 is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases19,20,21. O3 induces pulmonary inflammation and injury and is known to impair alveolar macrophage phagocytosis of bacterial pathogens22,23. However, it is unknown whether O3 impairs alveolar macrophage efferocytosis. Investigating O3-induced alterations in alveolar macrophage efferocytosis will provide potential insight into how exposure can lead to chronic pulmonary disease incidence and exacerbation. Described below is a simple method to evaluate alveolar macrophage efferocytosis in the lungs of female mice after acute O3 exposure.
The outlined method posseses several advantages over other efferocytosis protocols commonly used in the field by eliminating the use of costly fluorescent dyes, extensive flow cytometry measurements, and ex vivo manipulation of alveolar macrophages24,25. Additionally, this protocol measures alveolar macrophage efferocytosis in the context of the lung microenvironment, which can influence macrophage function.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Annexin V-FITC Kit</td>
			<td>Trevigen</td>
			<td>4830-250-K&#160;</td>
			<td>The TACS&#160;Annexin V-FITC Kit allows rapid, specific, and quantitative identification of apoptosis in individual cells when using flow cytometry.</td>
		</tr>
		<tr>
			<td>BCL2 Jurkat T Cells&#160;</td>
			<td>ATCC</td>
			<td>ATCC CRL-2899</td>
			<td>The BCL2 Jurkat cell line was derived by transfecting human Jurkat T cells with the pSFFV-neo mammalian expression vector containing the human BCL-2 ORF insert and a neomycin-resistant gene. Has been for models of measuring efferocytosis.&#160;</td>
		</tr>
		<tr>
			<td>Countess II Automated Cell Counter</td>
			<td>Thermofisher</td>
			<td>AMQAX1000</td>
			<td>It is a benchtop assay platform equipped with state-of-the-art optics, full autofocus, and image analysis software for rapid assessment of cells in suspension. Very easy to use.</td>
		</tr>
		<tr>
			<td>Cytospin 4 Cytocentrifuge</td>
			<td>Thermofisher</td>
			<td>A78300003</td>
			<td>Provides economical thin-layer preparations from any liquid matrix, especially hypocellular fluids such as bronchoalveolar lavage fluid.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, heat inactivated</td>
			<td>Thermofisher</td>
			<td>16140071</td>
			<td>Provides Nutrients to cultured cells for them to grow. It is standard for cell culture.&#160;</td>
		</tr>
		<tr>
			<td>Kwik-Diff&#160; Reagent 2, Eosin</td>
			<td>Thermofisher</td>
			<td>9990706</td>
			<td>Eosin staining that stains cytoplasm.</td>
		</tr>
		<tr>
			<td>Kwik-Diff Reagent 1, Fixative</td>
			<td>Thermofisher</td>
			<td>9990705</td>
			<td>Fixes cells to be stained by H&#38;E.</td>
		</tr>
		<tr>
			<td>Kwik-Diff Reagent 3, Methylene Blue</td>
			<td>Thermofisher</td>
			<td>9990707</td>
			<td>Methylene Blue staining that stains the nucleus.</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma/Aldrich</td>
			<td>P0781-100ML</td>
			<td>Penicillin-Streptomycin is the most commonly used antibiotic solution for culture of mammalian cells. Additionally it is used to maintain sterile conditions during cell culture.</td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium, GlutaMAX Supplement&#160;</td>
			<td>Thermofisher</td>
			<td>61870036</td>
			<td>RPMI 1640 Medium (Roswell Park Memorial Institute 1640 Medium) was originally developed to culture human leukemic cells in suspension and as a monolayer. RPMI 1640 medium has since been found suitable for a variety of mammalian cells, including HeLa, Jurkat, MCF-7, PC12, PBMC, astrocytes, and carcinomas. Helps grow Jurkat T cells fast and efficiently.</td>
		</tr>
		<tr>
			<td>Stratagene UV Stratalinker 1800 UV Crosslinker</td>
			<td>Cambridge Scientific&#160;</td>
			<td>16659</td>
			<td>The Stratalinker UV crosslinker is designed to induce apoptosis, crosslink DNA or RNA to nylon, nitrocellulose, or nylon-reinforced nitrocellulose membranes.</td>
		</tr>
		<tr>
			<td>Teledyne T400 ultraviolet light photometer&#160;</td>
			<td>Teledyne API</td>
			<td>T400</td>
			<td>The Model T400 UV Absorption analyzer uses a system based on the Beer-Lambert law for measuring low ranges of ozone in ambient air.</td>
		</tr>
		<tr>
			<td>Teledyne T703 Ozone calibrator</td>
			<td>Teledyne API</td>
			<td>T703</td>
			<td>Provides feedback control of the UV lamp intensity, assuring stable ozone output.</td>
		</tr>
	</tbody>
</table>

in vivo assessment of alveolar macrophage efferocytosis following ozone exposure

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

O3 exposure is known to induce pulmonary inflammation and injury, and efferocytosis is required to maintain tissue homeostasis. C57BL/6J female mice were exposed to filtered air (FA) or 1 ppm O3 for 3 h and necropsied 24 h post-exposure to examine pulmonary inflammation and injury. O3-exposed mice displayed a significant increase in macrophages and neutrophils in the airspace compared to the FA control group (Figure 1A...

Watch this Scientific Journal Video about In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure at JoVE.com

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

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

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

in-vivo-assessment-alveolar-macrophage-efferocytosis-following-ozone

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JoVE Journal

Immunology and Infection

9.0K Views.  East Carolina University. This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis.

Video: In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of malaria.

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

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

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a promising future of using miRNAs in the diagnosis and care of lung diseases. Here, we define a protocol for miRNA profiling to measure the relative abundance of a group of miRNAs predicted to regulate inflammatory genes in the lung tissue from of an ozone-induced airway inflammation mouse model. Because it has been shown that circulating sex hormone levels can affect the regulation of lung innate immunity in females, the purpose of this method is to describe an inflammatory miRNA profiling protocol in female mice, taking into consideration the estrous cycle stage of each animal at the time of ozone exposure. We also address applicable bioinformatics approaches to miRNA discovery and target identification methods using limma, an R/Bioconductor software, and functional analysis software to understand the biological context and pathways associated with differential miRNA expression.

Here we describe a method to assess lung expression of miRNAs that are predicted to regulate inflammatory genes using mice exposed to ozone or filtered air at different stages of the estrous cycle.

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a ...

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

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

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

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

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

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Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice