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2 protocols using apc cy7 conjugated anti b220

1

Multiparameter Immune Cell Profiling

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APC-conjugated anti-CD11c, anti-CD44, and anti–IFN-γ mAbs; APC-Cy7–conjugated anti-B220, anti-CD4, and anti-CD11c mAbs; FITC-conjugated anti–MHC class II mAb; PB-conjugated anti-CD11c, anti-CD45, and anti–MHC class II mAbs; PE-conjugated anti-CD8, anti-CD62L, and anti-TCRβ mAbs; PE-Cy7–conjugated anti-CD45 and anti–MHC class II mAbs; and annexin V and PI were purchased from eBioscience. BLT1 antagonist U-75302 was purchased from Cayman Chemical. A ChemR23 agonist (recombinant murine chemerin) was purchased from R&D Systems. BLT1 antagonist ONO-4057 was provided by Ono Pharmaceutical Co., Ltd. RvE1 was synthesized as previously described (Ogawa and Kobayashi, 2009 (link)).
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2

Multiparametric Flow Cytometry Assay

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Cell suspensions were incubated with fluorochrome labeled-Abs for 30 min at 4°C. Different combinations of the following anti-mouse Abs (BD Biosciences, eBioscience, Biolegend) were used: PerCP-Cy5.5-conjugated anti-CD4, FITC-conjugated anti-CD8 and anti-CD3, PE-conjugated anti-CD25, APCconjugated anti-gd-TCR and anti-CD19, PE-Cy7-conjugated anti-NK1.1, APC-Cy7-conjugated anti-B220, PE-conjugated anti-PD-1, anti-PD-L1 and anti-PD-L2. Transcription factors Forhead box P3 (FoxP3) and interferon-regulatory factor-4 (IRF-4) were detected after cell fixation and permeabilization with FoxP3 Staining Buffer Set (eBioscience) using the following antibodies: APC-conjugated anti-FoxP3 and eFluor660conjugated anti-IRF4 (eBioscience). For intracellular cytokine staining, DCs or allogenic cultures were exposed to brefeldin A (10 μg/ml; Sigma) for the last 4 h of cell culture. Cells were fixed and permeabilized with Cytofix/Cytoperm and Perm/Wash (BD Biosciences) and incubated with surface staining antibodies and PE-conjugated anti-IL-17 and APC-conjugated anti-IFN-g (Biolegend). Cells were acquired on BD FACSCanto II and analyzed using the FlowJo software.
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