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

Take cytometer tubes containing a staining mixture comprising fluorescently labeled monoclonal antibodies suspended in a stimulation buffer.

Add decreasing concentrations of the test allergen into several tubes. Add anti-IgE antibodies to one tube to serve as the IgE-mediated positive control, and add buffer to another tube for the negative control.

Pipette human peripheral blood into the tubes. Incubate.

If the individual is sensitized to the test allergen, this exposure causes the allergen to bind to the allergen-specific IgE antibodies bound to Fc-receptors on the basophil surface.

Allergens crosslink IgE-bound Fc-receptors, causing basophil activation.

Basophils undergo degranulation, releasing intracellular granules containing inflammatory mediators, with the upregulation of CD63 and CD203c molecules.

Fluorescently-labeled monoclonal antibodies bind to CD63, CD203c, and CCR3 on basophils, respectively.

Lower the temperature to stop basophil degranulation.

Add buffer to lyse erythrocytes and fix the cells.

Using a flow cytometer, identify the basophils from the lymphocyte population.

Using CD63 as an activation marker, analyze basophil activation at decreasing allergen concentrations.

Begin by collecting peripheral blood in 9-milliliter heparinized tubes, and place the samples in a rotor to maintain them at room temperature until they are required for the experimental protocol.

Label two 5-milliliter cytometer tubes each for negative control and positive control. Also, label one tube each for the different allergen or drug concentrations being tested. Then, place the tubes in a rack so that the tubes fit perfectly without slipping.

For the first positive control, prepare a 4 micromolar solution of N-Formylmethionyl-leucyl-phenylalanine, or fMLP, in 0.05% PBST to confirm the quality of basophils. For the second positive control, prepare a 0.05 milligrams per milliliter anti-IgE solution using PBST. Prepare the staining mix by adding 1 microliter each of CCR3-APC, CD203c-PE, and CD63-FITC antibodies per 20 microliters of the stimulation buffer. Then, add 23 microliters of this staining mix to each tube.

Add 100 microliters of PBST to the negative control tubes, add 100 microliters of fMLP to one of the positive control tubes, and add 100 microliters of anti-IgE to the other. To the rest of the tubes, add 100 microliters of the different allergen or drug concentrations, and incubate at 37 degrees Celsius for 10 minutes. After incubation, add 100 microliters of blood to each tube gently to avoid hemolysis. Then, gently vortex the tubes and incubate them for 25 minutes at 37 degrees Celsius in the thermostatic bath with medium agitation.

Keep the tubes at 4 degrees Celsius for at least five minutes to stop the degranulation. To each tube, add 2 milliliters of lysing buffer to lyse the erythrocytes. Vortex each tube, and then, incubate the tubes for 5 minutes at room temperature. Centrifuge the tubes at 300 x g at 4 degrees Celsius for 5 minutes. Then, overturn the rack into the sink to decant the supernatant while the cells remain at the bottom of the tubes. Add 3 milliliters of PBST to each tube to wash the cells, and vortex the tubes. Perform a second round of centrifugation using the same set of parameters and decant the supernatant by overturning the rack into the sink. Then, keep the samples at 4 degrees Celsius, protected from light until flow cytometry.

Connect the flow cytometer to the computer software and wait until the cytometer is ready. Load the template and instrument settings as described in the text manuscript. Start the process of sample acquisition.

To select activated basophils, first, get the lymphocytes from the Side Scatter - Forward Scatter plot. Then get the basophils from the lymphocyte population as CCR3-positive CD203c-positive cells, and acquire at least 500 basophils per tube. Set the CD63-negative threshold to approximately 2.5% using the negative control tubes, and analyze the samples.