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.
All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Sample preparation
2. Staining mix preparation
3. Blood stimulation
4. Erythrocytes lysing
5. Flow cytometry acquisition
Table 1: Flow cytometer requirements
Light Sources (lasers) | 488 nm Coherent SapphireTM air-cooled argon-ion laser; 20 mW; 633-nm JDS UniphaseTM HeNe air-cooled laser; 17 mW |
Light excitatory wavelength | Blue laser:488 nm; Red laser: 633 nm |
Light source power at the excitatory wavelength | Blue laser: 20 mW; Red laser: 17 mW |
Optical filters | SSC: 488/10; FITC: 530/30, PE:585/42, APC: 660/20 |
Optical detectors | FSC, SSC, FL1-H FITC, FL2-H PE, FL4-H APC |
Optical detectors tye | Air-cooled argon-ion laser |
Optical paths | BD octagon (488 nm laser line); BD Trigons (633 nm laser line) |
The authors have nothing to disclose.
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 |