Violetdye

The CD8⁺ T cells were isolated from
Rag^−/− OT-1 TCR transgenic mice using a CD8
isolation kit (Miltenyi Biotec, Auburn, CA). The CD44⁺ cells
were depleted using biotinylated anti-mouse CD44 (1:2000 dilution, BioLegend,
San Diego, CA) and anti-biotin beads (Miltenyi Biotec). The naïve
CD8⁺ OT-1 T cells were labeled with either CFSE or violet
dye (both from Invitrogen, Grand Island, NY) at 5 μM. Ten microliters of
CFSE-labeled cells (1×10⁸ ml⁻¹) were
directly injected into the vaginal tissue (Ivag) using a Hamilton syringe with a
32G needle. One hundred microliters of violet dye-labeled cells
(1×10⁷ ml⁻¹) were injected into the
tail vein (IV). The mice were under isoflurane general anesthesia during the
performance of dual transfer.

Brains of neonatal mice were dissected to remove pial membrane and minced into 1-2mm fragments in cold PBS. Then tissues were dissociated in Type IV collagenase (400U/ml, Worthington) and DNase I (32U/ml, Worthington) in PBS with Ca2⁺ and Mg2⁺ (Hyclone) at 37 °C on a rotator for 30 min. After disaggregation, cells were washed in cold PBS and passed through a 40-μm filter followed by centrifugation for 5min at 500g. Supernatant was discarded and cell pellet was resuspended in 20%BSA and centrifuged at 1000g for 15min to remove the myelin. The red blood cells were lysed with RBC lysis buffer. Afterwards, the cells were washed in PBS and stained with the Violet Dye (Invitrogen, L34955) to exclude dead cells according to the manufacture’s instruction. After washing in PBS, the cells were blocked with CD16/32 (eBioscience, 14-0161) and stained with CD31-APC (eBioscience, 17-0311) to label endothelial cells at 4°C for 30 min. The stained cells were analyzed using a FACS Aria II Flow Cytometer (BD Bioscience).