Cd11b pe tr

Bone marrow was flushed out of femurs and tibias of Mpzl3 −/− or +/+ littermates (6 weeks old). The bone marrow was labeled using biotinylated antibodies against CD3, CD4, CD8 and B220 (eBioscience, San Diego, CA). Streptavidin-labeled microbeads were then used to magnetically deplete the biotinylated lymphocytes using autoMACS (Milteyni Biotec, Inc., Auburn, CA). 3 × 10⁶ bone marrow cells after depletion were injected intraperitoneally into 2-day old B6 Rag −/− mice (The Jackson Laboratory, Bar Harbor, ME). After 10 weeks, the inguinal lymph nodes and spleens of these mice were analyzed by flow cytometry, and skin analyzed by histology. For flow cytometry, 3 × 10⁶ cells were blocked using a cocktail of anti-CD16/32 (2.4G2) and normal mouse sera (Jackson ImmunoResearch, West Grove, PA) before being stained with fluorescently labeled antibodies to determine their phenotype and activation status. The following antibody conjugates were used: CD11b PE-TR (Life Technologies Corp.), CD4 V500 (Becton Dickinson, San Jose, CA), CD11c FITC, Gr-1 PECy7, CD3 Alexa Fluor 700, CD8 efluor605, B220 PerCpCy5.5, CD44 efluor450, CD62L APC, CD69 PE (eBioscience). Cells were then washed and re-suspended in 2% FBS in PBS for analysis with flow cytometers (LSR-II and Fortessa, Becton Dickinson).