Facscanto plus

Apoptotic cells were analyzed by flow cytometry after staining with FITC-Annexin V and propidium iodide (cat no. 640914; BioLegend, Inc.). Briefly, cells were washed twice with cold Cell Staining buffer, and then resuspended in Annexin V Binding buffer at a concentration of 5x10⁶ cells/ml. Add 5 µl of FITC-Annexin V and 10 µl of propidium iodide solution into 100 µl of cell suspension, and then the cells were gently vortexed and incubated for 15 min at 25˚C in the dark. Add 400 µl of Annexin V Binding Buffer to each tube and then the cells were subjected to flow cytometry analysis using FACSCanto Plus instrument (BD Biosciences). The results were analyzed using FlowJo software (v.10.4.1; Tree Star, Inc.).

Flow cytometry was performed on a FACSCanto Plus instrument (BD Biosciences) and FlowJo v.10 (FlowJo, LLC) was used for data analysis.
Antibody list: APC-conjugated CD3 antibody (BD Pharmingen 555340); V450-conjugated CD4 antibody (BD Pharmingen 560650); PE-Cy7-conjugated CD8 antibody (BD Pharmingen 560960); FITC-labeled goat anti-mouse IgG(H+L) antibody (Sigma); FITC-conjugated IL13Ra antibody (BD Biosciences); and APC-labeled anti-CD107a-APC antibody (BD Biosciences).

BAT is used for the in vitro diagnosis of IgE-mediated allergies. C-C chemokine receptor (CCR) 3 and cluster of differentiation (CD) 63 expressions have been considered as an indicator of basophil activation for measuring specific IgE.22 (link)23 (link) Basically, peripheral blood samples from cat-allergic volunteers were collected on EDTA within 24 hours. Leukocytes were isolated by means of red blood cell lysis and washed twice with PBS. Later, the cells were stimulated with 100 µL of activation buffer supplemented with either anti-IgE or rCat-NPC2 (final concentration, 10 µg/mL) at 37°C for 30 minutes. The stimulation was stopped by adding 900 µL of cold PBS containing 2.5 mM EDTA, followed by centrifugation at 4°C for 5 minutes. After removal of the supernatant, the pellets were resuspended with 100 µL of staining cocktail containing 5 µL of anti-human Alexa647-conjugated CD63 antibody (561983, BD Biosciences, Frankin Lakes, NJ, USA) and 5 µL of phycoerythrin-conjugated CCR3 antibody (184123, eBioscience Inc., San Diego, CA, USA) and thereafter incubated for 20 minutes on ice. Basophil expressions of CCR3 and CD63 were evaluated using a FACSCanto Plus and analyzed using the build-in Diva software (BD Biosciences).