The authors would like to thank Katrin Bossmann, Anett Neumann and Eike Wolter (German Environment Agency) for their valuable preparatory work.

NOTE: This protocol describes the indirect staining of PM10 particles with a monoclonal antibody (monoclonal mouse IgG1 antibody, clone MA-3B4) against Bet v 1, the major birch pollen antigen component, plus an Allophycocyanin (APC)-labeled secondary antibody (anti-Mouse IgG1 antibody, clone A85-1) and the subsequent analysis on a flow cytometer. With appropriate other antibodies available, this method might be extended to the detection of other antigens bound to ambient air particles.
1. PM10 Sampling
<ol>
	<li>Collect PM10 from ambient air on polytetrafluoroethylene (PTFE) filters using a low volume sampler with a flow rate of 2.3 m3/hr (Figure 1). A characterization of the sampler used for the experiments described here is found in16. Running time depends on the amount of PM10 needed (usually between 1 and 10 days).</li>
	<li>At the end of the incubation time, remove the filter from the sampler and freeze it at -20 &#176;C until use.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/54721/54721fig1.jpg" /> 
Figure 1. Low volume PM10 sampler. Example of a low volume PM10 sampler. <a href="http://cloudfront.jove.com/files/ftp_upload/54721/54721fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. PM10 Removal and Particle Count
<ol>
	<li>Let the PTFE filter thaw for about 5 min. Then, put the filter in a clean polystyrol Petri dish (Figure 2A). Take a new Petri dish for each filter, if more than one filter is processed.</li>
	<li>Subsequently, overlay the PTFE filter with phosphate-buffered saline (PBS). This protocol is established for a final PM10 concentration of 8x106 particles per ml (see step 3.3). To obtain at least that concentration, use the following empirical volume of PBS to overlay the filter with: If the PM10 collection time was &#60;2 days, use 2 ml. For incubation times &#8805;2 days, use 4 ml. 
	NOTE: In order to increase the particle concentration of the PM10 suspension, suspensions from different filters can be pooled, if this is appropriate.</li>
	<li>Hold the PTFE filter with tweezers and brush with an electrical toothbrush with a sensitive brush head for 1 min (Figures 2B, 2C). Transfer the particle-PBS-suspension, hereafter termed PM10 suspension, to a clean reaction tube.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/54721/54721fig2.jpg" /> 
Figure 2. PM10 removal with an electrical toothbrush. A polytetrafluoroethylene filter with sampled PM10 is placed in a polystyrol Petri dish (A) and is overlaid with 4 ml PBS. Then, PM10 is removed with an electrical toothbrush (B: before brushing and C: after brushing for 1 min). <a href="http://cloudfront.jove.com/files/ftp_upload/54721/54721fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Measure the concentration of PM10 particles, e.g., by use of a particle counter. Dilute an adequate volume of PM10 suspension such as&#160;50 &#181;l in 10 ml isotonic measurement buffer, measure three times and calculate the mean total number of particles per ml. Be sure to use a particle counter which can detect particles of the relevant size.</li>
</ol>
3. Bet v 1 Staining
<ol>
	<li>Calculate the number of reaction tubes required for analysis of the sample: At least three reaction tubes are needed: (i) one tube with sample only (native control) (ii) one tube with sample plus secondary antibody (negative control), and (iii) one tube with sample plus primary and secondary antibody (specific sample). If measured for the first time, prepare at least two reaction tubes for (i) (ii), and (iii) to have enough material to properly adjust cytometer settings (see step 4.1).</li>
	<li>Calculate the amount of PM10 suspension required for the number of reaction tubes. Each reaction tube requires 50 &#181;l of suspension.</li>
	<li>Adjust the particle concentration measured in step 2.4 for the volume of suspension calculated in step 3.2 to a final concentration of 8x106 particles per ml by adding an appropriate amount of PBS. 
	NOTE: The remaining PM10 suspension may be frozen at -20 &#176;C for future experiments, although for this protocol freshly prepared PM10 suspension is recommended.</li>
	<li>Block unspecific binding by supplementing the PM10 suspension with bovine serum albumin (BSA) at a final concentration of 0.02%, using a stock solution such as 1% BSA prepared with PBS. Vortex briefly and incubate for 20 min at room temperature.</li>
	<li>For each sample to be analyzed by flow cytometry, transfer 50 &#181;l of the suspension from step 3.4 to a clean reaction tube. Add a monoclonal mouse antibody against Bet v 1 at a final concentration of 0.02 &#181;g/&#181;l to the reaction tubes designated for specific staining, vortex briefly and incubate for 60 min at room temperature.
	<ol>
		<li>Let the reaction tubes with the PM10-BSA suspension designated for the native control and the negative control also stay for 60 min at room temperature.</li>
	</ol>
	</li>
	<li>Wash all samples by adding 500 &#181;l PBS supplemented with 0.02% BSA to each reaction tube, vortex briefly and centrifuge the samples subsequently at 4,700 x g for 5 min at room temperature. Discard the supernatant carefully by using a vacuum pump.</li>
	<li>Repeat step 3.6.</li>
	<li>Determine the total amount of APC-labeled secondary anti-Mouse IgG1 antibody needed: 1 &#181;g antibody dissolved in 50 &#181;l PBS supplemented with 0.02% BSA per reaction tube. Dilute the appropriate quantity of antibody with the respective volume of PBS supplemented with 0.02% BSA.</li>
	<li>Add 50 &#181;l of diluted secondary antibody to all reaction tubes except for the reaction tube designated for the native control. Supplement the latter with 50 &#181;l PBS supplemented with 0.02% BSA. Vortex all samples for a few seconds.</li>
	<li>Incubate all samples for 30 min in the dark.</li>
	<li>Repeat step 3.6 twice.</li>
	<li>Add 50 &#181;l PBS to each reaction tube, vortex and analyze the samples on a flow cytometer.</li>
</ol>
4. Flow Cytometric Analysis
<ol>
	<li>Using the native control, adjust the following cytometer parameters to optimize data analysis.
	<ol>
		<li>By using the forward scatter (FSC) threshold controller displayed on the device control board, set the FSC threshold at the lowest value (200) and start the analysis.</li>
		<li>By using the scatter voltage controller, adjust the FSC and sideward scatter (SSC) in that way that all PM10 particles can be detected and that the population of particles is located approximately in the middle of the FSC axis and in the lower half of the SSC axis.</li>
		<li>By using the fluorescence voltage controller, adjust the APC voltage and another fluorescence voltage like fluorescein isothiocyanate (FITC), which does not emit at the same wave length like APC, if necessary. Make sure, that all particles are visible in the lower half of both fluorescence axes.</li>
	</ol>
	</li>
	<li>Consecutively, examine each sample including the native control and store the FSC, SSC, APC, and FITC data of at least 10,000 particles per sample.</li>
	<li>Evaluate the data with the respective software. Adsorbed allergen content in the specific sample can be quantified in two ways:
	<ol>
		<li>Analyze the APC fluorescence intensity of all particles (median value) as a measure of Bet v 1 load over all PM10 particles. 
		NOTE: If the specific sample contained allergen, APC fluorescence intensity should increase in the specific sample compared to the negative control. In contrast, other fluorescence intensities (e.g., FITC fluorescence intensity) should not significantly change.</li>
		<li>Calculate the percentage of PM10 particles with bound anti-Bet v 1 antibody. Thereby, in the negative control, set a gate around the particles considered APC positive, copy and paste this gate into the specific sample and subtract the percentage of APC positive particles in the negative control from the percentage of APC positive particles in the specific sample.</li>
	</ol>
	</li>
</ol>

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Y., 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=The+association+of+ambient+air+pollution+with+airway+inflammation+in+schoolchildren.">The association of ambient air pollution with airway inflammation in schoolchildren.</a> Am J Epidemiol. 175 (8), 764-774 (2012).</li><li>Cyprowski, M., Buczynska, A., Szadkowska-Stanczyk, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indoor+allergens+in+settled+dust+from+kindergartens+in+city+of+Lodz,+Poland.">Indoor allergens in settled dust from kindergartens in city of Lodz, Poland.</a> Int J Occup Med Environ Health. 26 (6), 890-899 (2013).</li><li>Brough, H. 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=Distribution+of+peanut+protein+in+the+home+environment.">Distribution of peanut protein in the home environment.</a> J Allergy Clin Immunol. 132 (3), 623-629 (2013).</li><li>Galli, S. J., Tsai, M., Piliponsky, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+allergic+inflammation.">The development of allergic inflammation.</a> Nature. 454 (7203), 445-454 (2008).</li><li> World Health Organisation, R. O. f. 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Swiss Study on Air Pollution and Lung Diseases in Adults.</a> Int Arch Allergy Immunol. 106 (2), 149-156 (1995).</li><li>Grote, M., Valenta, R., Reichelt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abortive+pollen+germination:+a+mechanism+of+allergen+release+in+birch,+alder,+and+hazel+revealed+by+immunogold+electron+microscopy.">Abortive pollen germination: a mechanism of allergen release in birch, alder, and hazel revealed by immunogold electron microscopy.</a> J Allergy Clin Immunol. 111 (5), 1017-1023 (2003).</li><li>Namork, E., Johansen, B. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentrations+of+the+major+birch+tree+allergen+Bet+v+1+in+pollen+and+respirable+fine+particles+in+the+atmosphere.">Concentrations of the major birch tree allergen Bet v 1 in pollen and respirable fine particles in the atmosphere.</a> J Allergy Clin Immunol. 100 (5), 656-661 (1997).</li><li>Buters, J. T., 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=The+allergen+Bet+v+1+in+fractions+of+ambient+air+deviates+from+birch+pollen+counts.">The allergen Bet v 1 in fractions of ambient air deviates from birch pollen counts.</a> Allergy. 65 (7), 850-858 (2010).</li><li>Buters, J. T. M., 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=Release+of+Bet+v+1+from+birch+pollen+from+5+European+countries.+Results+from+the+HIALINE+study.">Release of Bet v 1 from birch pollen from 5 European countries. Results from the HIALINE study.</a> Atmos Environ. 55, 496-505 (2012).</li><li>Ormstad, H., Johansen, B. V., Gaarder, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airborne+house+dust+particles+and+diesel+exhaust+particles+as+allergen+carriers.">Airborne house dust particles and diesel exhaust particles as allergen carriers.</a> Clin Exp Allergy. 28 (6), 702-708 (1998).</li><li>Brough, H. 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The authors have nothing to disclose.

A critical step of the protocol is the use of an appropriate filter for the collection of PM10 particles from ambient air (see step 1.1). The filter has to be strong enough to endure brushing with an electrical toothbrush, and not all filter materials fulfill this requirement. The staining protocol was established with a PM10 particle concentration of 8x106 particles per ml. However if the material is limited and pooling of samples is not appropriate, the method will probably function as well, but the antibody concentrations (see steps 3.5 and 3.8) might have to be adjusted.
Bet v 1 staining of PM10 particles did not result in distinct populations of positively and negatively stained particles. This might be caused by the varying amounts of Bet v 1 allergen adsorbed to each of the particles ranging from very little up to a high amount. This could result in the expansion of the APC signal thus shifting the population towards APC positivity. As it is difficult to separate the positive from the negative particles, two quantification methods were used to determine the differences in the Bet v 1 content of the PM10&#62;0.5 specimens: (i) relative quantification by measuring the median APC fluorescence intensity of all particles, and (ii) determining the percentage of APC positive particles. Regarding the Bet v 1 load of particles from low and high pollen season PM10&#62;0.5, both methods revealed similar results. Still, relative quantification by median fluorescence intensity of all particles is recommended as it is independent of placing the gate and therefore probably less error-prone.
To date many studies examine the allergen content in ambient air particulate matter by extracting the respective allergen and subsequent quantification with ELISA5,12-14,17. There is a fundamental difference between the procedure described here and the quantification with ELISA: ELISA quantifies the extracted and dissolved antigen, while flow cytometry analyzes the particle-bound antigen. By means of ELISA the Bet v 1 load of the tested PM10 samples (n=8) was below the detection limit of 1.2 ng/ml (data not shown). Similarly, Buters and others identified no Bet v 1 in the PM &#60;2.5 &#181;m fraction and only about 7% in the 10 &#181;m &#62;PM &#62;2.5 &#181;m fraction, but more than 93% in the PM &#62;10 &#181;m fraction of ambient air13. The contrasting results of the ELISA on the one hand and the FACS analysis on the other hand, may be caused by differences in the detection method in conjunction with divergent sensitivity. Further research however is needed to fully understand this difference.
A method to visualize particle-bound antigen is scanning electron microscopy10,14. By scanning electron microscopy, Ormstad et al. visualized Bet v 1 on the surface of suspended particulate matter soot particles sampled in the high pollen season and to a lesser extent on particles sampled in the low pollen season15. Additionally, allergens from pollen, latex and also &#946;-glucans were found to be adsorbed to combustion particles in ambient air10. This method, however, does not allow quantification of the particle-bound allergen.
By use of flow cytometry, particle-bound Bet v 1 allergen could be quantified. Thus, flow cytometry may offer a new way to characterize the 10 to 0.5 &#181;m biological fraction of PM10 as with other suitable antibodies on hand, this method might be extended to the detection of other antigens on ambient air particles,&#160;e.g., mold, dust mite allergens or LPS. As PM10 particles adsorb not only biological material, but also chemicals and metals quite easily, unspecific binding of antibodies could, however, pose a problem. If a new antibody is tested, a critical step is to prove specific binding. This can be done by, for example, blocking the binding capacity of the specific antibody with the corresponding antigen prior to staining11.
As Bet v 1 content of ambient air can differ from birch pollen count12,13,18, allergic symptoms might perhaps correlate better with allergen level than with pollen count14,18. Hence, the presented method in conjunction with clinical data enables to examine in future experiments whether allergic reactions to birch correspond to the Bet v 1 allergen load of PM10&#62;0.5.

Air pollution is being considered as an important environmental cause of the increased incidence and severity of respiratory allergies observed in recent decades1-3. Moreover, there was growing interest in the distribution of common allergens in dust4,5.
Birch pollen can provoke hay fever but can also be an important trigger of allergic asthma6-8. Whole birch pollen is not likely to enter the lower respiratory tract or to be found in PM10 as a result of its size (22 &#181;m in diameter). However, birch pollen allergens like Bet v 1, the major birch pollen allergen component, can be released after pollen rupture9 and can bind to ambient air particles10, thus possibly enter the lower respiratory airways. Indeed, it has been shown that PM10 may contain biologically active allergens as demonstrated by in vitro activation of basophiles from a pollen allergic proband11.
Bet v 1 allergen content in PM10 samples has been studied by extracting the respective allergen and subsequent quantification with ELISA12-14. With the ELISA technique, the dissolved allergen was measured, but the amount of allergen-loaded particles still remained unknown. Scanning electron microscopy revealed allergen-loaded particles but did not allow quantification10,15.
This study employs flow cytometry to quantify the proportion of Bet v 1-loaded PM10 particles in ambient air samples. Due to the detection limit of the flow cytometer only particles larger than 0.5 &#181;m can be examined. The &#62;0.5 &#181;m fraction of PM10 will be further referred to as PM10&#62;0.5.

<table><tbody><tr><td>Teflon filter</td><td>Pall Life Sciences, USA</td><td>R2PL047</td><td>47 mm, 1.0 &amp;micro;m</td></tr><tr><td>low volume sampler</td><td>Sven Leckel Ingenieur B&amp;uuml;ro GmbH, Germany</td><td>LVS3</td><td>air flow of 2.3 m&lt;sup&gt;3&lt;/sup&gt;/hr</td></tr><tr><td>Phosphate-buffered saline</td><td>Biochrom, Germany</td><td>L1825</td><td>without Ca/Mg, low endotoxin</td></tr><tr><td>electrical toothbrush&amp;nbsp;</td><td>Braun, Germany</td><td>Oral-B Vitality Sensitive</td><td></td></tr><tr><td>Casy cell counter&amp;nbsp;</td><td>Sch&amp;auml;rfe System GmbH, Germany</td><td>Model TTC</td><td>range of detectable particle size: 0.7 &amp;micro;m to 45&amp;nbsp;&amp;micro;m</td></tr><tr><td>FACSCanto II&amp;nbsp;</td><td>Becton Dickinson, USA</td><td>3-laser, 8-color (4-2-2)</td><td></td></tr><tr><td>FACS Diva Software v6.1.3&amp;nbsp;</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>bovine serum albumin (BSA)&amp;nbsp;</td><td>Sigma-Aldrich, USA</td><td>A2153-10G</td><td></td></tr><tr><td>monoclonal mouse IgG1 antibody against Bet v 1</td><td>Indoor Biotechnologies, UK</td><td>&amp;nbsp;MA-3B4</td><td>clone MA-3B4</td></tr><tr><td>APC (Allophycocyanin)-labeled secondary anti-Mouse IgG1 antibody&amp;nbsp;</td><td>Becton Dickinson</td><td>560089</td><td>clone A85-1</td></tr><tr><td>SPSSTM software version 18&amp;nbsp;</td><td>PASW Statistics 18, Hongkong, China</td><td></td><td></td></tr><tr><td>Petri Dish</td><td>Gosselin, France</td><td>BP50-02</td><td>D 55&amp;nbsp;mm, H 15&amp;nbsp;mm</td></tr><tr><td>FACS Tube&amp;nbsp;</td><td>Becton Dickinson, USA</td><td>REF 352054</td><td>5 ml Polystyrene</td></tr><tr><td>CASYton</td><td>Roche&lt;br/&gt; Germany</td><td>REF 05651808001</td><td></td></tr><tr><td>Matrix Blank Tubes</td><td>Thermo Scientific, USA</td><td>4140</td><td>1.4 ml, PP</td></tr><tr><td>Centrifuge</td><td>Heraeus, Thermo Scientific</td><td>Megafuge 40R</td><td></td></tr><tr><td>Vacuum Pump</td><td>INTEGRA Biosciences AG, Switzerland</td><td>Model 158 320</td><td>Inetrgra Vacusafe</td></tr><tr><td>recombinant Bet v 1a antigen&amp;nbsp;</td><td>Indoor Biotechnologies, UK</td><td>LTR-BV1A-1</td><td>Concentration: 2.0 mg/ml</td></tr></tbody></table>

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.

Bet v 1 allergen adsorption to PM10&#62;0.5 particles was quantified by indirect antibody staining and subsequent analysis on a flow cytometer. A PM10 sample from high pollen season served as template. As stated in step 3.1, the negative control consisted of PM10 particles incubated with APC labeled secondary antibody only (Figure 3A). PM10 particles stained with anti-Bet v 1 antibody plus secondary antibody displayed the allergen loaded particles (Figure 3B). As described in step 4.3, two ways of quantifying the allergen load were used: On the one hand, the median value of the APC fluorescence intensity of all particles was analyzed being 137 for the negative control and 904 for the specific sample. On the other hand, the percentage of particles with bound anti-Bet v 1 antibody was determined: A gate was set around the APC positive particles in the negative control and subsequently copied and pasted into the specific sample. In the negative control, 3% of the PM10&#62;0.5 particles were considered APC positive. This percentage of false positive particles was subtracted from the percentage of positive particles in the specific sample thus resulting in 77.5% APC positive PM10&#62;0.5 particles in the specific sample. To prove that the observed binding of the anti-Bet v 1 antibody was specific, binding capacity was blocked with the corresponding antigen prior to staining. This diminished binding of the anti-Bet v 1 antibody by 69%, if quantified by APC fluorescence intensity, and by 84%, if quantified by percentage of Bet v 1 positive PM10&#62;0.5 particles (Figure 3C).
<img alt="Figure 3" src="/files/ftp_upload/54721/54721fig3.jpg" /> 
Figure 3. Particle-bound Bet v 1 allergen can be visualized by flow cytometry. APC fluorescence intensity of a PM10 sample from high pollen season stained only with the APC labeled secondary antibody (A), stained with the anti-Bet v 1 primary antibody and subsequently with the APC labeled secondary antibody (B), and after blocking the primary antibody with recombinant Bet v 1 antigen (C). The gate PAPC+ was set around the particles considered APC positive (displayed in red) and respective percentages are given. This figure has been slightly modified from11. <a href="http://cloudflare.jove.com/files/ftp_upload/54721/54721fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To test whether this method could be adopted to reveal differences in the amount of adsorbed Bet v 1 content of PM10&#62;0.5 samples from high and from low pollen season, 13 PM10 samples from high and 6 PM10 samples from low pollen season were analyzed. Figure 4 depicts significant differences in the APC fluorescence intensity and in the proportion of Bet v 1 positive PM10&#62;0.5 particles of PM10 samples from high pollen season compared to PM10 samples from low pollen season. Both quantification methods hereby showed similar results.
<img alt="Figure 4" src="/files/ftp_upload/54721/54721fig4.jpg" /> 
Figure 4. PM10 samples from low and high pollen season differ in their amount of adsorbed Bet v 1. Low pollen season PM10 was sampled in autumn/winter 2013 (n=6), high pollen season PM10 in May 2012 and 2013 (n=13). (A) The APC fluorescence intensity of PM10&#62;0.5 particles from high pollen season was significantly higher than from low pollen season (median/min/max high pollen season: 796/313/1097; median/min/max low pollen season: 197/85/277). (B) PM10 from high pollen season contained significantly more Bet v 1-positive PM10&#62;0.5 particles than PM10 from low pollen season (median/min/max high pollen season: 45.2/18.5/74.5; median/min/max low pollen season: 11.8/4.4/19.8). Box plots show median values (inner line of the box), 25. and 75. percentiles, respectively (lower and upper borders of the box) and minimum and maximum values (whiskers). ***p&#60;0.001 versus low pollen season, Mann Whitney U test. This figure has been slightly modified from11. <a href="http://cloudflare.jove.com/files/ftp_upload/54721/54721fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10 at JoVE.com

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

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 ...

flow-cytometric-analysis-of-particle-bound-bet-v-1-allergen-in-pm10

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

7.5K Views.  Federal Environment Agency. 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 >0.5 µm in diameter, can be used to measure these allergen-loaded particles.

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

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

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

Environment

Visualizing Field Data Collection Procedures of Exposure and Biomarker Assessments for the Household Air Pollution Intervention Network Trial in India

Here, we present a visual representation of standard procedures to collect population-level data on personal exposures to household air pollution (HAP) from two different study sites in a resource-constrained setting of Tamil Nadu, India. Particulate matter PM2.5 (particles smaller than 2.5 microns in aerodynamic diameter), carbon monoxide (CO), and black carbon (BC) were measured in pregnant mothers (M), other adult women (OAW), and children (C) at various times over a 4 year period. In addition, stove usage monitoring (SUMs) with data-logging thermometers and ambient measurements of air pollution were carried out. Furthermore, the feasibility of collecting biological samples (urine and dried blood spots [DBSs]) from study participants at the field sites was successfully demonstrated. Based on findings from this and earlier studies, the methods used here have enhanced the data quality and avoided issues with household air pollution and biological sample collection in resource-constrained situations. The procedures established may be a valuable educational tool and resource for researchers conducting similar air pollution and health studies in India and other low- and middle-income countries (LMICs).

We detail the consistent, high-quality procedures used throughout air and biological sampling processes at Indian field sites during a large randomized controlled trial. Insights gathered from the oversight of applications of innovative technologies, adapted for exposure assessment in rural regions, enable better field data collection practices with more reliable outcomes.

Here, we present a visual representation of standard procedures to collect population-level data on personal exposures to household air pollution (HAP) ...

Quantifying Carbon in Pulmonary Macrophages via Induced Sputum Analysis

Measuring Carbon Content in Airway Macrophages Exposed to Carbon-Containing Particulate Matters

Pulmonary macrophages exhibit a dose-dependent pattern in phagocytizing particles. Following engulfment, these macrophages are subsequently excreted with sputum, rendering macrophages and particles visible and quantifiable under light microscopy. Notably, elemental carbon within the mammalian body originates exclusively from external contaminants. Consequently, the carbon content in airway macrophages (CCAM) serves as a valid exposure biomarker, accurately estimating individual exposure to carbon-containing particulate matter (PM). This article delineates a protocol involving sputum collection, preservation, processing, slide preparation, and staining, as well as macrophage photo acquisition and analysis. After removing the macrophage nuclei, the proportion of cytoplasm area occupied by carbon particles (PCOC) was calculated to quantify carbon content in each macrophage. The results indicate an elevation in CCAM levels after exposure to carbon-containing PM. In summary, this non-invasive, precise, reliable, and standardized method enables the direct measurement of carbon particles within target cells and is utilized for large-scale quantification of individual CCAM through induced sputum.

This article describes a detailed experimental protocol for measuring the carbon content of airway macrophages with the aim of assessing the internal exposure dose at the level of individual particulate matter exposure.

Pulmonary macrophages exhibit a dose-dependent pattern in phagocytizing particles. Following engulfment, these macrophages are subsequently excreted with ...

A Component-resolved Diagnostic Approach for a Study on Grass Pollen Allergens in Chinese Southerners with Allergic Rhinitis and/or Asthma

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen allergens, data on this topic are limited in southern China. Any available data were obtained by self-report questionnaires, skin prick tests, and total or specific IgE tests using crude extracts. For many reasons, these methods are unreliable. Serum sIgE reactivity to Bermuda grass, Timothy grass, and Humulus scandens allergens in a cohort of patients from Greater Guangzhou (southern China&#39;s largest city and its outskirts) with allergic rhinitis and/or asthma were examined using a fully-automated immunoassay analyzer as a component-resolved diagnostics (CRD) tool.
For the first time, a considerably high prevalence of Bermuda grass sIgE positivity was demonstrated in Chinese southerners with allergic rhinitis and/or asthma. In these patients, a subtle prevalence of sensitization to Timothy grass and Humulus scandens was also noted, which may arise from cross-reactivity, as the latter two are not common in the region. This was also supported by the detection of allergen components.
Fully-automated immunoassay analyzers may offer satisfactory consistency between regions, laboratories, and institutions and over time. The automaticity of the instrument may enable a standardized detection that would not have been readily revealed before the advent of CRD.
This is a study that uses a CRD approach to investigate sensitization to grass pollen allergens in southern China. It adds to current evidence in the literature. Future studies are needed to validate these findings. However, although CRD is a useful tool, the findings made with the fully-automated immunoassay analyzer should not substitute for other laboratory investigations, clinical evaluations, and physician expertise.

This work describes a protocol that uses a component-resolved approach to study sensitization to grass pollen allergens in a cohort of patients from southern China with allergic rhinitis and/or asthma.

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen ...

Medicine

Identification and Characterization of Immunogenic RNA Species in HDM Allergens that Modulate Eosinophilic Lung Inflammation

Environmental allergens such as house dust mites (HDM) are often in complex forms containing both allergic proteins that drive aberrant type 2 responses and microbial substances that induce innate immune responses. These allergen-associated microbial components play an important role in regulating the development of type 2 inflammatory conditions such as allergic asthma. However, the underlying mechanisms remain largely undefined. The protocol presented here determines the structural characteristics and in vivo activity of allergen-associated immunostimulatory RNA. Specifically, common allergens are examined for the presence of double-stranded RNA (dsRNA) species that can stimulate IFN responses in lungs and restrain&#160;the development of severe lung eosinophilia in a mouse model of HDM-induced allergic asthma. Here, we have included the following three assays: Dot blot to show the dsRNA structures in total RNA isolated from allergens including HDM species, RT-qPCR to measure the activities of HDM RNA in interferon stimulating genes (ISGs) expression in mouse lungs and FACS analysis to determine the effects of HDM RNA on the number of eosinophils in BAL and lung, respectively.

Environmental allergens such as house dust mites (HDM) often contain microbial substances that activate innate immune responses to regulate allergic inflammation. The protocol presented here demonstrates the identification of dsRNA species in HDM allergens and characterization of their immunogenic activities in modulating eosinophilic lung inflammation.

Environmental allergens such as house dust mites (HDM) are often in complex forms containing both allergic proteins that drive aberrant type 2 responses ...

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

Humanized Mediator Release Assay as a Read-Out for Allergen Potency

Mediator release assays analyze in vitro immunoglobulin E (IgE)-mediated degranulation and secretion of mediators by effector cells, such as mast cells and basophils, upon stimulation with serial dilutions of putative allergens. Therefore, these assays represent an essential tool that mimics the in vivo degranulation process, which occurs upon allergen exposure in sensitized patients or in skin prick tests. Additionally, these assays are usually employed to investigate the allergenic potential of proteins and the reactivity of patients' sera's reactivity. Herein, we describe a simple 2-day protocol using an immortalized rat basophil leukemia cell line transfected and humanized with the human high-affinity IgE plasma-membrane receptor (Fc&#949;RI). This variant of the mediator release assay is a robust, sensitive, and reproducible in vitro cell-based system without the need to immobilize the antigen to solid matrices. The protocol consists of the following steps: (1) complement inactivation of human sera, (2) harvesting, seeding, and passive sensitization of the cells, (3) stimulation with antigen to cause mediator release, and (4) measuring of &#946;-hexosaminidase activity as a surrogate for the released inflammatory mediators, such as histamine. The assay represents a useful tool to assess the capacity of the allergen-IgE cross-linking to trigger cell degranulation and can be implemented to standardize allergen extracts, to compare patients' reactivity to minor or major allergens and to allergenic extracts (pollen, cat dander, etc.), to investigate the potency of allergen homologs, isoforms, and fold-variants (e.g., hypoallergenicity), as well as the effects of ligands on the allergenic activity. A more recent application includes the use of the assay to monitor the treatment efficacy in the course of allergen immunotherapy.

Here we present the mediator release assay, using a rat basophilic leukemia cell line transfected with the human IgE receptor, to simulate the degranulation of effector cells typically observed in type 1 allergic reactions. This method investigates the biological activity of allergens in a highly sensitive, reproducible, and tailorable manner.

Mediator release assays analyze in vitro immunoglobulin E (IgE)-mediated degranulation and secretion of mediators by effector cells, such as mast cells ...