Double staining of intracellular cytokine proteins and T-lymphocyte subsets. Evaluation of the method in blood and bronchoalveolar lavage fluid

被引:6
作者
de Pater-Huijsen, FL
van der Loos, CM
de Riemer, MJ
van der Zee, JS
Jansen, HM
Out, TA
机构
[1] Univ Amsterdam, Acad Med Ctr, Dept Pulm, NL-1105 AZ Amsterdam, Netherlands
[2] Univ Amsterdam, Acad Med Ctr, Clin & Lab Immunol Unit, NL-1105 AZ Amsterdam, Netherlands
[3] CLB Sanquin Blood Supply Fdn, Expt & Clin Immunol Lab, Amsterdam, Netherlands
来源
HISTOCHEMICAL JOURNAL | 2000年 / 32卷 / 01期
关键词
D O I
10.1023/A:1003967726621
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
An immunocytochemical staining method has been developed for simultaneous staining of both cell surface markers (CD4 and CD8) and intracellular cytokine proteins IFN-gamma, IL-4 and IL-5. Cell surface molecules were visualized with alkaline phosphatase, which was developed by Fast Blue BB. Intracellular cytokine proteins were detected by amino-ethyl carbazole. We applied this technique to T cells from T-cell lines and T-cell clones, peripheral blood mononuclear cells and broncho-alveolar lavage fluid cells. Cells were used either unstimulated or stimulated for 4 h with 1 ng/ml PMA and 1 mu g/ml ionomycin, which proved to be an optimal stimulus taking cytokine staining, cell recovery and cell viability into account. We studied peripheral blood mononuclear cells from healthy subjects and found that without in vitro stimulation on average 0.4% of the cells were IFN-gamma positive cells. In unstimulated broncho-alveolar lavage fluid cells of the 2 allergic asthmatic subjects studied so far we found higher numbers of cytokine-positive cells (up to 22% of the lymphocytes being IL-4(+) cells). By in vitro stimulation, the numbers of cytokine-positive peripheral blood mononuclear cells from the healthy subjects were increased to maximally 5% IFN-gamma(+) cells. In stimulated lavage fluid cells from allergic asthmatic subjects maximally 34% of the lymphocytes became IFN-gamma(+). We conclude that this method allows detection of intracellular cytokine proteins in both CD4(+) and CD8(+) T cells without the need for stimulating the cells in vitro. In vitro stimulation may change the cytokine profile detected.
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页码:3 / 11
页数:9
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