期刊
JOURNAL OF IMMUNOLOGICAL METHODS
卷 359, 期 1-2, 页码 56-60出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jim.2010.05.002
关键词
Tissue lysate; Functional assays; CFSE; Autoimmunity
资金
- Australian National Health and Medical Research Council (NHMRC) [559007]
- Juvenile Diabetes Research Foundation [JDRF 4-2006-1025]
- Victorian Government
Currently human T-cell responses to unpurified tissue extracts cannot be easily measured because current cell lysis methods yield a lysate that is toxic. Here we describe the optimization of a protocol for extracting proteins from human tissues in a format that is compatible with the functional analysis of human T cells. The tissue was homogenized in a mixture of butan-1-ol, acetonitrile and water and then lyophilized. Lyophilized protein extracts were dissolved in 8 M urea because urea did not affect T-cell function when present at 0.08 M or less. Using this method cytokine production and proliferation responses were detected from islet, acinar and spleen extracts. Hence, our method allows the rapid preparation of human tissue lysates in a format that is compatible with the analysis T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity. (C) 2010 Elsevier B.V. All rights reserved.
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