Digested organs were

further stained with biotinylated-anti-4–1BBL ex vivo, followed by Streptavidin-PE or Streptavidin-allophycocyanin amplification. Biotinylated anti-4–1BBL-treated 4–1BBL-deficient mice were used as a negative staining control for analysis of 4–1BBL expression. The samples were analyzed using FACScalibur, FACSCanto, or LSR II (BD Biosciences) with Cell-Quest or FACSDiva acquisition software. Data analysis was done using FlowJo software (TreeStar Inc., Ashland, OR, USA). Marrow was flushed from the femurs and tibias of 10 C57BL/6 mice and digested for 45 min at 37°C with 0.2 mg/mL Collagenase P (Roche) and 0.2 mg/mL DNase I (Sigma). The single cells were seeded in Petri dishes at a density Selleckchem C59 wnt of 1–2 × 106 cells/cm2. One day later, nonadherent cells were washed away and the remaining adherent cells were expanded in medium (see Generation of memory T cells in vitro) for up to 25 days. Half of the medium was replaced with fresh medium once a week. On day 25, adherent cells were removed by trypsinization, stained with anti-CD45.2 (ebioscience), anti-VCAM-1 (ebioscience),

biotinylated anti-4–1BBL (19H3), and secondary streptavidin, and assessed by flow cytometry. In addition, CD45-negative cells were sorted into the VCAM-1+ and VCAM-1− population. Yields of CD45−VCAM-1+ cells were approximately 500,000 in all three experiments, whereas yields of CD45−VCAM-1− cells ranged 13,000- 165,000 cells. Total RNA was extracted and purified using RNeasy Micro kit (Qiagen). Random mTOR inhibitor hexamer primers and purified RNA were used for the reverse transcription reaction (Invitrogen). PCR of the cDNA was done using following primers: 4–1BBL forward: 5′-CTT GAT GTG GAG GAT ACC-3′, 4–1BBL reverse: 5′-GCT TGG

CGA ACA CAG GAG-3′, CCL19 forward: 5′-GCC TCA GAT TAT CTG CCA T-3′, CCL19 reverse: 5′-AGA CAC AGG GCT CCT TCT GGT-3′, IL-7 forward: 5′-TCC TCC ACT GAT CCT TGT TC-3′, IL-7 reverse: 5′-TTG TGT GCC TTG TGA TAC TG-3′, CXCL12 forward: 5′-GTC CTC TTG CTG TCC AGC TC-3′, CXCL12 reverse: 5′-TAA TTT CGG GTC AAT GCA CA-3′, actin forward: 5′-GGG AAT GGG TCA GAA GGA-3′, actin reverse: 5′-AAG AAG GAA GGC TGG AAA-3′, GAPDH forward: 5′-AAC TTT GGC ATT GTG GAA GG-3′, and GAPDH reverse: 5′-GGA ASK1 GAC AAC CTG GTC CTC AG-3′. CD8+ memory+ T cells were generated in vitro from OT-I DsRed splenocytes as described [29]. A total of 6 × 106 cells were adoptively transferred into C57BL/6 mice. One day later, femurs were harvested, fixed for 4 h in 4% paraformaldehyde at 4°C, dehydrated in 10, 20, and 30% sucrose solution for 24 h, respectively, and frozen in SCEM embedding medium (Section-Lab Co. Ltd., Yokohama, Japan). Bone cryosections (7 μm) were prepared using Kawamoto’s Film Method [51]. Sections were stained with the following Ab from eBioscience if not indicated otherwise: VCAM-1 (429), CD31 (MEC13.