In Vivo Tracking of Intestinal IgA+ B Cells

For migration imaging, IgA-Cre/YC3.60^flox mice (8–12 weeks-old) were anesthetized with a mixture of three types of anesthetic agents as described previously (19 (link)). An incision was carefully made in the abdominal wall, and the small intestine was exposed. PPs were identified by naked eyes. About 8000 sorted CD11b⁺IgA⁺ PP B cells and 30 000 CD11b⁻IgA⁺ PP B cells were labeled with CellTracker Orange CMTMR fluorescent dye (Invitrogen, USA) and then injected to a PP of an IgA-Cre/YC3.60^flox transgenic mouse directly by a 25 μl syringe (Trajan Scientific and Medical, Australia). The PP with transferred cells was observed under an LSM 880 microscope (Carl Zeiss, Germany). Images were analyzed with ZEN2009 software (Carl Zeiss, Germany). After one-hour observation of the PP under a microscope, the abdominal incision was carefully closed with an ELP Skin Stapler (Akiyama Co. Ltd. Japan). Forty hours after transfer, under anesthesia, the PP with transferred cells was observed again to identify the localization of transferred CD11b⁺IgA⁺ PP B cells under an LSM 880 microscope (Carl Zeiss, Germany) and analyzed with ZEN2009 software (Carl Zeiss, Germany).