Anti cd11b pe cy5

Mouse gp100_25–33 (mgp100, EGSRNQDWL) and human gp100_25–33 (hgp100, KVPRNQDWL) peptides were synthesized by Peptron (Daejeon, Korea). CD8 microbeads were purchased from Miltenyi Biotec (Auburn, AL, USA). All antibodies used for flow cytometry were purchased from BD Bioscience, including anti-Thy1.1-FITC, anti-CD3-FITC, anti-B220-FITC, anti-CD62L-FITC, anti-Ly-6C-FITC, anti-CD19-PE, anti-CD8-PE, anti-CD44-PE, anti-CD8-PE-Cy5, anti-Thy1.1-PE-Cy5, anti-CD11b-PE-Cy5, anti-CD4-APC, anti-CD8-APC, and anti-CD45-APC. Recombinant human IL-2 (Proleukin) was purchased from Novartis, and the Fc fusion protein of human IL-7 (IL7-Fc), which consist of the extracellular domain of human IL-7 (aa 26–177) fused to the N-terminus of the Fc portion of a mutant human IgG1, was obtained from AdipoGen (Seoul, Korea). The CellTrace CFSE Cell Proliferation Kit was purchased from Invitrogen (Carlsbad, CA, USA).

Splenocytes were purified as described above and were re-suspended in FACS buffer (1% FBS in PBS). TLR2 was detected by monoclonal anti-TLR2-FITC (Imegenex, IMG-6320C). Ficoll-purified bone marrow of WT control mice and Dek-KO mice or whole blood samples was re-suspended in 1% BSA and 1% horse serum in PBS. Samples were spun at 1,600 rpm for 5 min at 4 °C. Cell pellets were re-suspended with anti-Ly6G-FITC and or anti-CD11b–PE-Cy5 (BD Pharmingen, #553312) antibodies and incubated on ice for 30 min. Isotype-matched IgGs were used as negative control antibodies. Samples were centrifuged at 1,600 rpm for 5 min at 4 °C and fixed with 2% paraformaldehyde. Cell surface markers were analysed by FACS.