Genome-wide analysis of bone marrow-derived dendritic cells, unstimulated or stimulated with Bifidobacterium in vitro.

Comparison of gene expression in bone marrow-derived dendritic cells (DCs) unstimulated or stimulated with Bifidobacterium in vitro. BMDC stimulation with Bifidobacterium in vitro: Cells isolated from the tibiae and femurs of WT C57BL/6 mice were cultured in DMEM medium containing 10% FBS and 1% penicillin/streptomycin, in the presence of rmGM-CSF (20 ng/ml; BioLegend) for 8 days at 37°C with 5% CO2. BMDCs were then stimulated for 4 hours with medium alone or with rehydrated Bifidobacterium-containing medium at a ratio of 1:10 BMDCs to bacterial cells. Total RNA was isolated using RNeasy® Micro kit (Qiagen) and submitted to the Functional Genomics Facility at the University of Chicago for gene expression profiling. RNA integrity and concentration were assessed using an AgilentBioanalyzer 2100, and all RNA samples used for microarray analysis had an RNA Integrity Number = 10.0.

体外未刺激或经双歧杆菌（Bifidobacterium）刺激的骨髓源树突状细胞（DCs）的基因表达比较研究。体外双歧杆菌刺激骨髓源树突状细胞的实验方案如下：从野生型（WT）C57BL/6小鼠的胫骨与股骨中分离细胞，在含有10%胎牛血清（FBS）、1%青霉素/链霉素的达尔伯克改良伊格尔培养基（DMEM）中，添加重组小鼠粒细胞-巨噬细胞集落刺激因子（rmGM-CSF，20 ng/ml；BioLegend），于37℃、5% CO₂条件下培养8天。随后将骨髓源树突状细胞分别以仅含基础培养基的培养液，或按骨髓源树突状细胞与细菌细胞数量比1:10配制的复水含双歧杆菌培养基刺激4小时。使用RNeasy® Micro试剂盒（Qiagen公司）提取总RNA，并将样本送至芝加哥大学功能基因组学中心开展基因表达谱分析。采用安捷伦生物分析仪2100（AgilentBioanalyzer 2100）评估RNA的完整性与浓度，所有用于微阵列分析的RNA样本的RNA完整性指数（RIN）均为10.0。