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