Analyzing Basophil Activation in the Presence of a Test Allergen using Flow Cytometry
Published: November 30, 2023
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 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. Label 5 mL cytometer tubes for negative controls (2 tubes), positive controls (2 tubes), and different concentrations of allergen/drug (1 t…
開示
The authors have nothing to disclose.
Materials
| 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 |
