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

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

1. 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).
2. At the end of the incubation time, remove the filter from the sampler and freeze it at -20 °C until use.

Figure 1. Low volume PM10 sampler. Example of a low volume PM10 sampler. Please click here to view a larger version of this figure.

2. PM10 Removal and Particle Count

1. 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.
2. Subsequently, overlay the PTFE filter with phosphate-buffered saline (PBS). This protocol is established for a final PM10 concentration of 8×10⁶ 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 <2 days, use 2 ml. For incubation times ≥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.
3. 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.

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). Please click here to view a larger version of this figure.

4. Measure the concentration of PM10 particles, e.g., by use of a particle counter. Dilute an adequate volume of PM10 suspension such as 50 µl 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.

3. Bet v 1 Staining

1. 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).
2. Calculate the amount of PM10 suspension required for the number of reaction tubes. Each reaction tube requires 50 µl of suspension.
3. Adjust the particle concentration measured in step 2.4 for the volume of suspension calculated in step 3.2 to a final concentration of 8×10⁶ particles per ml by adding an appropriate amount of PBS.
   NOTE: The remaining PM10 suspension may be frozen at -20 °C for future experiments, although for this protocol freshly prepared PM10 suspension is recommended.
4. 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.
5. For each sample to be analyzed by flow cytometry, transfer 50 µl 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 µg/µl to the reaction tubes designated for specific staining, vortex briefly and incubate for 60 min at room temperature.
   1. 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.
6. Wash all samples by adding 500 µl 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.
7. Repeat step 3.6.
8. Determine the total amount of APC-labeled secondary anti-Mouse IgG1 antibody needed: 1 µg antibody dissolved in 50 µl 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.
9. Add 50 µl of diluted secondary antibody to all reaction tubes except for the reaction tube designated for the native control. Supplement the latter with 50 µl PBS supplemented with 0.02% BSA. Vortex all samples for a few seconds.
10. Incubate all samples for 30 min in the dark.
11. Repeat step 3.6 twice.
12. Add 50 µl PBS to each reaction tube, vortex and analyze the samples on a flow cytometer.

4. Flow Cytometric Analysis

1. Using the native control, adjust the following cytometer parameters to optimize data analysis.
   1. 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.
   2. 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.
   3. 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.
2. 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.
3. Evaluate the data with the respective software. Adsorbed allergen content in the specific sample can be quantified in two ways:
   1. 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.
   2. 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.