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

Abstract

Source: Doña, I. et al., Basophil Activation Test for Allergy Diagnosis. J. Vis. Exp. (2021)

In this video, we demonstrate the test to evaluate basophil activation in the presence of a test allergen using flow cytometry.

Protocol

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 buff....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5 mL Round Bottom Polystyrene Test Tube, without Cap, Nonsterile	Corning	352008
APC anti-human CD193 (CCR3) Antibody	BioLegend	310708
BD FACSCalibur Flow Cytometer	BD Biosciences
Calcium chloride	Sigma-Aldrich	C1016
FITC anti-human CD63 Antibody	BioLegend	353006
HEPES (1 M)	Thermo-Fisher	15630106
Lysing Solution 10x concentrated	BD Biosciences	349202
Magnesium chloride	Sigma-Aldrich	M8266
N-Formyl-Met-Leu-Phe	Sigma-Aldrich	F3506
PE anti-human CD203c (E-NPP3) Antibody	BioLegend	324606
Potassium chloride	Sigma-Aldrich	P9541
Purified Mouse Anti-Human IgE	BD Biosciences	555857
Recombinant Human IL-3	R&D Systems	203-IL
Sheath Fluid	BD Biosciences	342003
Sodium chloride	Sigma-Aldrich	S3014
TUBE 9 mL LH Lithium Heparin	Greiner Bio-One	455084
Tween 20	Sigma-Aldrich	P1379