Sample collection
To determine the antibody (Ab) responses, the mice were bled a week
after immunization and the serum was collected. Saliva sample was
collected with pipetting method. Saliva samples harvested that PBS
placed in the oral cavity at 3 minutes later, transferred to a
microcentrifuge tube. For intestinal IgA, the large intestine was
excised 5 cm (from the rectum), The saliva and, intestinal fluid from
the entire gut was collected, mixed with cold saline (4 °C) and
centrifuged for 10 min at 2000 ×g. The supernatants were collected and
frozen. Peritoneal exudate cells were collected by intraperitoneal
injection with 10 mL of DMEM medium. The cells were centrifuged at 2500
rpm for 10 min and the supernatants were discarded. These were
resuspended in DMEM complete medium. Splenocytes were prepared from the
spleens by chopping on sterile glass sides and centrifugation at 1400
rpm for 5 min. The erythrocytes were lysed with 1 mL of ammonium
chloride (0.8 % w/v). After centrifugation (1400 rpm for 5 min), 10 mL
of medium was added to the pelleted cells and filtered through a fine
cell strainer (BD Falcon, BD Bioscience) of 40 μm pore size. The cell
numbers and viability were assessed microscopically by the trypan blue
dye exclusion technique (Xu et al.,
2009).