Isolation of PBMCs
20ml peripheral blood would be collected from the patient before
provocation and 24h, 72h after allergen nasal challenge. Peripheral
blood was collected in the heparin anticoagulant according to a standard
manner, then transferred to a 50 mL centrifuge tube and diluted with 20
mL PBS. 2.5 mL Ficoll-Hypaque Solution (Meijingbio, China) were added to
the tube and mixed gently. Centrifuge at 400g for 25 minutes, and
transfer PBMC cell layer to another tube. Add enough HBBS and mix
thoroughly. After centrifuging at 300g for 5 minutes, the supernatant
was discarded and the cell pellet was re-suspended in HBSS to repeat the
centrifuging again.