Mice Lacking Expression of Secondary Lymphoid Organ Chemokine Have Defects in Lymphocyte Homing and Dendritic Cell Localization

Gunn, Michael D.; Kyuwa, Shigeru; Tam, Carmen; Kakiuchi, Terutaka; Matsuzawa, Akio; Williams, Lewis T.; Nakano, Hideki

Flow Cytometry. For DC quantitation, mesenteric, inguinal, axillary, and brachial LNs from four plt and two ϩ / ϩ mice were pooled, and single cell suspensions were prepared. RBCs were depleted by lysis, and cells at 5 ϫ 10 6 /ml were layered onto metrizamide (14.5 g/100 ml medium) and centrifuged for 10 min at 600 g . Cells at the interface were collected, washed, and resuspended in medium, stained with FITC-conjugated anti-I-Ad, and analyzed by flow cytometry on a FACScan ® (Becton Dickinson). Spleen cells were isolated as described previously (30) . In brief, spleens were minced, incubated for 1 h at 37 Њ C in RPMI with 130 U/ml collagenase and 0.1 mg/ml DNase, teased through 70-m nylon mesh, and centrifuged. RBCs were depleted by lysis, then cells were washed once and resuspended. Cells were stained with FITC-conjugated anti-I-Ad, biotinylated anti-B220, and biotinylated anti-CD3 followed by SA-PerCP and analyzed by flow cytometry. For DC quantitation, cells were gated against B220 and CD3 and analyzed for expression of I-Ad.