RESEARCH ARTICLE Sample preparation method for isolation of single-cell types from mouse liver for proteomic studies Wei Liu 1à , Yufang Hou 2à , Huahai Chen 3à , Handong Wei 1 , Weiran Lin 1 , Jichang Li 2 , Ming Zhang 3 , Fuchu He 1,4Ãà and Ying Jiang 1 1 State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, P. R. China 2 College of Veterinary Medicine, Northeast Agricultural University, Harbin, P. R. China 3 Guangxi Key Laboratory of Subtropical Bio-resource Conservation and Utilization, Animal Reproduction Institute, Guangxi University, Nanning, P. R. China 4 Institute of Biomedical Sciences, Fudan University, Shanghai, P. R. China Received: March 29, 2011 Revised: May 14, 2011 Accepted: June 8, 2011 It becomes increasingly clear that separation of pure cell populations provides a uniquely sensitive and accurate approach to protein profiling in biological systems and opens up a new area for proteomic analysis. The method we described could simultaneously isolate popula- tion of hepatocytes (HCs), hepatic stellate cells (HSCs), Kupffer cells (KCs) and liver sinu- soidal endothelial cells (LSECs) by a combination of collagenase-based density gradient centrifugation and magnetic activated cell sorting with high purity and yield for the first time. More than 98% of the isolated HCs were positive for cytokeratin 18, with a viability of 91%. Approximately 97% of the isolated HSCs expressed glial fibrillary acidic protein with a viability of 95%. Nearly 98% of isolated KCs expressed F4/80 with a viability of 94%. And the purity of LSECs reached up to 91% with a viability of 94%. And yield for HCs, HSCs, LSECs and KCs were 6.3, 1.3, 2.6 and 5.0 million per mouse. This systematic isolation method enables us to study the proteome profiling of different types of liver cells with high purity and yield, which is especially useful for sample preparation of Human Liver Proteome Project. Keywords: Hepatic stellate cells / Hepatocytes / Kupffer cells / Liver sinusoidal endothelial cells / Magnetic activated cell sorting / Technology 1 Introduction Current approaches to understanding the functional diversity of an organism preferentially strive for a systems approach whereby first the phenotypic classification of a specific cytome is achieved prior to an attempt to perform proteomic analysis. Proteomics research on tissue or organ worked on large populations of cells and thereby reported on population averages rather than their distributions missed rare (but important) events and has been unable to analyze cells that are only produced in small numbers. This is not by choice but owing to a paucity of techniques that allow experimentalists to measure proteome in individual cell types. Individual cell type proteome has become one of the most important fields of proteomics, and the primary cellular fractionation with high purity and yield has always been a challenge for cell biologists and also for the Human Liver Proteome Project. Abbreviations: Ac-LDL, acetylated low-density lipoprotein; ASGPR, asialoglycoprotein receptor; CK 18, cytokeratin 18; GBSS, Gey’s balanced salt solution; GFAP, glial fibrillary acidic protein; HC, hepatocyte; HSC, hepatic stellate cell; KC, Kupffer cell; LSEC, liver sinusoidal endothelial cell; MACS, magnetic activated cell sorting; NPC, nonparenchymal cell; PE, phycoerythrin; SEM, scanning electron microscopy; TEM, transmission electron microscopy à These authors contributed equally to this work. Ãà Additional corresponding author: Professor Fuchu He E-mail: hefc@nic.bmi.ac.cn Colour Online: See the article online to view Fig. 4 in colour. Correspondence: Dr. Ying Jiang, State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, P. R. China E-mail: jiangying304@hotmail.com Fax: 186-10-68177417 & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com 3556 Proteomics 2011, 11, 3556–3564 DOI 10.1002/pmic.201100157