Analyzing Basophil Activation in the Presence of a Test Allergen using Flow Cytometry

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Sample preparation

1. Collect peripheral blood in 9 mL heparinized tubes and maintain the sample at room temperature (RT) in a rotor until it is required for the experimental protocol.
2. Label 5 mL cytometer tubes for negative controls (2 tubes), positive controls (2 tubes), and different concentrations of allergen/drug (1 tube per each allergen/drug concentration tested). Place the tubes in a rack where tubes fit perfectly without slipping.
3. Prepare stimulation buffer in double distilled water containing 2% (v/v) HEPES, 78 mg/L NaCl, 3.7 mg/L KCl, 7.8 mg/L CaCl₂, 3.3 mg/L MgCl₂, 1 g/L HSA. Adjust pH to 7.4 and add IL-3 at 2 ng/mL. Usually, prepare 100 mL and divide into 2.5 mL aliquots to be frozen at -20 °C.
4. Prepare positive controls in PBS-Tween-20 0.05% (v/v) (PBS-T): Positive control 1, N-Formyl methionyl-leucyl-phenylalanine (fMLP) (4 µM), to confirm the quality of basophils; Positive control 2, Anti-IgE (0.05 mg/mL) as an IgE-mediated positive control.
5. Prepare the allergen/drug in PBS-T at 2x the desired final concentration.
   NOTE: Optimal allergen/drug concentrations to be used must be previously determined by using a wide range of concentrations, by dose-response curves, and by cytotoxicity studies following the same protocol steps.

2. Staining mix preparation

1. Add monoclonal antibodies labeled with fluorochrome to the stimulation buffer following the manufacturer's recommended antibody concentration or by previous antibody titration. In this protocol, we add 1 µL of each antibody (CCR3-APC and CD203c-PE for basophil identification; CD63-FITC for basophil activation) per 20 µL of stimulation buffer.
   NOTE: Protect the staining mix preparation from the light.
2. Add 23 µL of staining mix to each tube.

3. Blood stimulation

1. Add 100 µL of PBS-T to tubes 1 and 2 (negative control), 100 µL of fMLP to tube 3, 100 µL of anti-IgE to tube 4, and 100 µL of the different allergen/drug concentrations to the following tubes. Incubate for 10 min at 37 °C in a thermostatic bath with medium agitation to prewarm reagents.
2. Gently add 100 µL of blood to each tube to avoid hemolysis. Gently vortex tubes and incubate for 25 min at 37 °C in a thermostatic bath with medium agitation.
3. Stop the degranulation, keeping tubes at 4 °C for at least 5 min.
   NOTE: The protocol can be paused here at 4 °C for 30-45 min if required.

4. Erythrocytes lysing

1. Add 2 mL of 1x lysing buffer to each tube to lyse erythrocytes. Vortex each tube and incubate for 5 min at RT.
   NOTE: In this step, cells are fixed due to fixative agents (formaldehyde) contained in the buffer.
2. Centrifuge at 300 x g at 4 °C for 5 min. Decant the supernatant, overturning the rack into a sink. Cells remain at the bottom of the tubes.
3. Add 3 mL of PBS-T to each tube to wash cells. Vortex each tube.
4. Centrifuge at 300 x g at 4 °C for 5 min. Decant the supernatant, overturning the rack into a sink.
   NOTE: Keep samples at 4 °C, protected from the light until flow cytometer acquisition.

5. Flow cytometry acquisition

1. Acquire samples by flow cytometer (e.g., BD FACSCalibur Flow Cytometer). Connect the flow cytometer to the computer software and wait for the cytometer to be ready. Load the template and instrument settings (Table 1).
2. Start sample acquisition.
3. Use the following cytometer strategies for the selection of activated basophils.
   1. Gate the lymphocytes from the Side Scatter (SSC) – Forward Scatter (FSC) plot.
   2. Gate the basophils from the lymphocyte population as CCR3⁺CD203c⁺ cells. Acquire at least 500 basophils per tube.
   3. Show a CCR3 – CD63 plot to analyze activation using CD63 as an activation marker. Set the CD63 negative threshold to approximately 2.5% using the negative control tubes.
   4. Acquire all the samples.

Table 1: Flow cytometer requirements