Yin et al. BMC Genomics 2010, 11:612 http://www.biomedcentral.com/1471-2164/11/612 In-gel digestion was performed at 37°C overnight. The tryptic peptides were extracted from test gel pieces in 5 mL of 70% acetonitrile 5% HCOOH by sonication. The supernatant was dried under a Speed Vac and 6 μL of 1% HCOOH was then added to each test sample. Pro- tein identification using MALDI-Q-TOF-MS analysis was performed by the Proteomics Core Facility of the Institute of Biological Chemistry, Academia Sinica, Tai- wan [10]. The MS data with monoisotopic peptide masses were searched against the NCBI protein database using the MASCOT search engine (Matrix Science, London, UK). DC trafficking in vivo To analyze the migration of ex-vivo cultured DCs under in vivo experimental conditions in test mice, 1 × 106 cultured DCs were labeled with fluorescein isothiocyanate isomer I (See Figure 5; green fluorescence dye) at 300 μg/ mL at 37°C for 20 min, cells were then washed three times with PBS and subsequently injected into the tail vein of syngeneic BALB/c mice. Fresh lymph node (LN), spleen and liver tissues were harvested and frozen tissue sections of 12 μm in thickness were mounted on precleaned micro- scope slides (Superfrost/Plus; Fisher Scientific, Pittsburgh, PA), and stored at -80°C. To retain the fluorescent signal, the tissue sections were pretreated with 4% paraformalde- hyde at room temperature. Tissue sections were first incu- bated with blocking solution (2% fetal calf serum in PBS) for 10 min. Nuclear staining was then performed with 4,6- diamidino-2-phenylindole (DAPI) and followed by wash- ing with 1 × PBS (2 × 5 min each). Tissue sections were then mounted with 50% glycerol in H2O with coverslips. Fluorescence microscopy evaluation of immunostained frozen sections was performed using a Zeiss Axiovert 200 M microscope (Carl Zeiss, Heidelberg, Germany). Microscopy photos and images were captured with a digi- tal camera (Orca ER; Hamamatsu) and processed using Axiovision 4.6.3 (Carl Zeiss). The number of individually florescent spot as test cells was then scored for compara- tive analysis and the data was exported to Microsoft Excel. Pathway analysis of representative genes involved in/ associated with effects of [BF/S+L/Ep] on BMDCs Using a Web-based entry tool developed by Ingenuity Systems [46], findings presented in peer-reviewed scienti- fic publications were systematically encoded into ontol- ogy by content and modeling experts. A molecular network of direct physical, transcriptional and enzymatic interactions was observed between mammalian ortho- logs. For a better understanding of the temporal response of gene expression in the immune system, we con- structed a prototypical cell showing the possible candi- date signaling pathways, containing 35 genes (>2-fold change) responding to treatment with [BF/S+L/Ep]. The Page 17 of 18 candidate genes of interest also were keyed into TRANS- PATH professional database, version 7.1, (Biobase Biolo- gical Databases GmbH, Germany) to identify possible target molecules and signaling pathways, using four hier- archical levels of regulation [9,10]. Acknowledgements This work was supported by grants from the Genomic and Proteomic Program Academia Sinica (94F002-1), Taiwan, Republic of China. We thank Dr. Pei-Ing Huang, for her suggestions about the application of the bioinformatics software for cDNA microarray data, Ms. Ming-Yan Zhou for her technical assistance in performing cDNA microarray hybridized reaction, and Dr. Harry Wilson and Ms. Miranda Loney of Academia Sinica for professional editing of the manuscript. Author details 1Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan. 2Institute of Statistical Science, Academia Sinica, Taipei, Taiwan. 3Taiwan International Graduate Program (TIGP) - Molecular and Biological Agricultural Sciences Program, Academia Sinica, Taipei, Taiwan. 4Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan. 5Department of Mathematics, Tamkang University, Taipei County, Taiwan. Authors’ contributions SYY served as the key experimenter and author of the draft manuscript. WHW and KA provided useful revision and reorganization of the manuscript. 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