Name: in vivo assessment of alveolar macrophage efferocytosis following ozone exposure
Uploaded: 2019-10-22T13:00:00
Description: Watch this Scientific Journal Video about In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure at JoVE.com

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 ...

<ol>
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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...

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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 ...

The protocol described here measures lung macrophage efferocytosis after exposure to a criteria air pollutant ozone. These data indicate that ozone exposure decreases alveolar macrophage efferocytosis and thus could be a mechanism for why elevated levels of ozone increase the incidence of lung diseases. The main advantage of this technique is that it allows an in vivo assessment of alveolar macrophage efferocytosis without any ex vivo manipulations of the macrophages that may alter their phenotype or function.This technique has revealed a novel mechanism by which the lung cannot repair itself after air pollution exposure. Additionally, this technique may reveal novel pathways to targets that alter efferocytosis in the lung. This method can be applied to any model of lung injury or inflammation.However, the user will need to optimize when it is best to assess efferocytosis following lung injury. Performing the oropharyngeal aspiration can be technically challenging. I would advise working with your veterinary staff to make sure you have optimized your technique to perform this procedure.Begin by culturing Jurkat T cells in basal cell culture medium supplemented according to the manuscript directions at 37 degrees Celsius and 5%carbon dioxide. Jurkat T cells are a suspension cell line that can be maintained through passaging into pre-warmed culturing media every three days. Prepare apoptotic cells by growing them to 90%confluency which takes three to four days after passaging.Grow cells in five T75 flasks to obtain a sufficient number of cells for this protocol. Once the cells are ready, aspirate the flask contents about 24 milliliters and transfer them to a 50 milliliter conical tube. Count the cells using Trypan Blue staining and a hemocytometer.Pellet the cells by centrifugation at 271 times g for five minutes and discard the supernatant. Then, resuspend the cells in media to obtain three million cells per milliliter. Aliquot the cells into 100 by 20 millimeter tissue culture dishes by transferring five milliliters of cells per dish.Prepare approximately nine culture dishes. Set one dish aside as an unexposed control and expose the remaining dishes to UV.Set the UV crosslinker to the correct energy level by pressing the energy button and entering 600 with the number pad. Irradiate all dishes with the cells except for the control making sure to remove the top cover of the tissue culture dish during UV exposure.Then, incubate all the dishes in a cell culture incubator at 37 degrees Celsius and 5%carbon dioxide for four hours. After the incubation, combine all the irradiated cells into a 50 milliliter conical tube and pellet them by centrifugation at 271 times g for five minutes. Discard the supernatant and resuspend the cells in 24 milliliters of sterile PBS.Centrifuge the tube again and remove the supernatant. Prepare the cells for dosing mice by resuspending them in PBS at the appropriate concentration. Once the mouse is properly anesthetized, place it in a semi-recumbent supine position.Use surgical string tied between pegs on a slanted acrylic sheet board to suspend the mouse by the maxillary incisors. Use a pair of blunt non-ridged forceps to lightly grab and pull the mouse tongue and instill the apoptotic cells into the oral cavity with a P200 pipette. Dosing is successful when mice make a crackling noise one to two seconds after receiving the dose.With a gloved finger, gently cover the nose of the mouse until it inhales while the tongue is retracted. Keep the nose covered until no liquid is visible in the oral cavity and the mouse has taken two or more inhalations. Remove the mouse from the inoculation board and return it to the cage to allow it to recover from anesthesia making sure to place the mouse on its back to prevent debris from blocking the nares.After all of the mice have awoken from anesthesia, wait 90 minutes to allow alveolar macrophages to engulf influx of apoptotic cells. To collect lavage fluid, prepare the euthanized mouse according to the manuscript directions. Slowly push PBS into the lungs allowing them to inflate, then pull the same volume back out making sure the PBS is not exiting the nostrils.Collect the pooled lavage from each mouse in a 15 milliliter tube. Centrifuge the bronchoalveolar lavage or BAL at 610 times g for six minutes at four degrees Celsius, collect the supernatant into a 1.5 milliliter tube, and freeze it at minus 80 degrees Celsius. The pellet represents the cells from the bronchoalveolar space.Add one milliliter of ACK red blood cell lysis buffer to the pellet to remove the residual red blood cells. Vortex and lyse for one minute on ice, then add four milliliters of PBS to stop the lysis reaction. Centrifuge the cells at 610 times g for six minutes at four degrees Celsius and aspirate the supernatant with a vacuum aspirator.Then, resuspend the cells in one milliliter of PBS with 10%FBS and count the cells on a hemocytometer without Trypan Blue. Centrifuge 120 microliters of each sample onto slides at 12 times g for three minutes using medium acceleration and a cytocentrifuge. Leave the slides to dry overnight.The next day, stain the slides with hematoxylin and eosin in order to calculate both efferocytic and differential cell counts. Then, view slides under a Brightfield setting on a biological microscope using a 20X or 40X objective. Calculate the efferocytic index based on the ratio of alveolar macrophages that phagocytosed apoptotic Jurkat T cells to alveolar macrophages without apoptotic cell uptake making sure to count at least 200 cells from each slide.This protocol was used to investigate the effect of ozone exposure on pulmonary inflammation. Ozone exposed mice displayed an increase in macrophages and neutrophils in the air space compared to the filtered air control group and had an increase in BAL protein, a marker of alveolar epithelial dysfunction. Prior to dosing the mice, apoptosis in Jurkat T cells was confirmed with flow cytometry.The 60 milli joule UV exposure level and four-hour incubation time resulted in repetitive results of approximately 75%apoptotic cells. Efferocytic macrophages were identified as macrophages that had engulfed a Jurkat T cell compared to regular alveolar macrophages. There was a significant decrease in the efferocytic index of the ozone exposed group compared to the filtered air controls which indicates that ozone induced pulmonary inflammation is associated with decreased clearance of apoptotic cells.Attention to detail and writing down any observed differences is critical during this procedure. It is also recommended to blind the samples for analysis and have two different people count the macrophages. After isolation, the BAL cells can be used for mRNA analysis, stained for additional markers by immunofluorescence, and/or run on a flow cytometer to distinguish phenotypic changes.

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

Research

JoVE Journal

Immunology and Infection

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

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.