2.2 | Basophil activation test
Flow cytometric analysis based on the basophil activation test (BAT) was performed with an Allergenicity Kit (Beckman Coulter, Brea, CA) to measure CD203c.31 We added some reagents to measure additional parameters of CD63, surface-bound IgE, and FcεRI expressions. Whole blood samples for the BAT were collected from AD patients and HCs into blood collection tubes with ethylenediaminetetraacetic acid (EDTA). The BAT was performed within 24 hours of blood sampling.
Phosphate-buffered saline (PBS) was used as a negative control. Anti-IgE antibody (clone: E124-2-8D) from the Allergenicity Kit was used as a positive control (1 µg/mL) to stimulate basophils. PacificBlue-conjugated anti-human CD63 antibody (clone: H5C6; BioLegend, San Diego, CA) (0.9 µg/mL) was used to measure CD63 expression. VioBlue-conjugated anti-IgE antibody (clone: MB10-5C4; Miltenyi Biotec, Bergisch Gladbach, Germany) (0.5 µg/mL) was used to measure surface-bound IgE. Biotinylated anti-FcεRI antibody (clone: CRA1; Bio-Academia, Osaka, Japan) (11.2 µg/mL) was used to measure FcεRI expression. FcεRI expression corresponded with total FcεRI expression because the anti-FcεRI antibody used binds to the stalk region of the protein and does not inhibit IgE-binding.32 Anti-FcεRI antibody was also used as a stimulant for basophils. Basophil responsiveness to anti-FcεRI stimulation was measured.33 APC-Streptavidin (BD Biosciences, Franklin Lakes, NJ) (1.8 mg/mL) was used as a second-step reagent for the anti-FcεRI antibody.
Fifty milliliters of whole blood with EDTA, 10 µL of mixed staining reagent containing CRTH2-FITC, CD203c-PE, and CD3-PC7, and 50 µL of activation buffer were mixed in FACS tubes. Next, 10 µL of PBS as a negative control, 10 µL of anti-IgE antibody as a positive control, 0.5 µL of CD63, 0.6 µL of surface-bound IgE, or 1.25 µL of anti-FcεRI antibody was mixed into individual FACS tubes. The FACS tubes were incubated at 37℃ for 15 minutes. Biotinylated anti-FcεRI antibody was coupled with 1 µL of APC-Streptavidin as a second-step reagent at 4℃ for 30 minutes. Erythrocytes were depleted by adding fixative and lysis buffer for 10 minutes, followed by centrifugation at 200×g for 5 minutes. After removal of the supernatant, the cells were washed with 1500 µL of PBS, centrifuged at 200×g for 5 minutes, and fixed with 300 µL of 0.1% formaldehyde. Basophil samples were measured by flow cytometry (FACS Verse; BD Biosciences, San Jose, CA). The flow cytometry data were analyzed with FlowJo software (BD Biosciences, Franklin Lakes, NJ).
For basophil detection and characterization, we employed forward scatter (FSC), side scatter (SSC), and fluorochromes (CRTH2-FITC, CD203c-PE, anti-FcεRI antibody-APC, CD63 and surface-bound IgE-Pacific blue, and CD3-PC7). Histograms were created for FSC and SSC to eliminate red blood cell debris and select the total leucocyte population. CD3-positive T-lymphocytes were eliminated by PC7. Basophils were selected as the CRTH2-positive/CD203c-positive/CD3-negative population. Basophil activation was detected as the CD203c/CD63-high population. Mean fluorescence intensity (MFI) was measured. Based on the MFI, CD203c and CD63 expression under the condition with no stimulation (PBS) were defined as ‘baseline MFI’, those under the condition with anti-IgE antibody stimulation were defined as ‘anti-IgE stimulation MFI’, and those under the condition with anti-FcεRI antibody stimulation were defined as ‘anti-FcεRI stimulation MFI’.34 To calculate the responsiveness of basophils, we divided stimulation MFI by baseline MFI and presented it as the ‘response ratio’. The gating technique is shown in the supplemental material (Figure S1).