IMPACT OF ENZYMATIC TISSUE DISINTEGRATION ON THE LEVEL OF SURFACE MOLECULE EXPRESSION AND IMMUNE CELL FUNCTION
IMPACT OF ENZYMATIC TISSUE DISINTEGRATION ON THE LEVEL OF SURFACE MOLECULE EXPRESSION AND IMMUNE CELL FUNCTION
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DOI：
10.1556/eujmi.2.2012.2.3
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发表时间：
2012-06-01
影响因子：
2.2
通讯作者：
Bruder, D.
中科院分区：
文献类型：
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作者：
Autengruber, A.;Gereke, M.;Bruder, D.
对存在于特定解剖部位的免疫细胞进行免疫学特性分析，通常需要基于酶解组织的方法将其从相应组织中分离出来。我们对原代脾细胞进行酶消化处理，评估了胶原酶和分散酶（这两种酶常用于从组织中释放免疫细胞）对48种免疫相关表面分子可检测性的影响，这些分子常用于流式细胞术鉴定、分离和表征免疫细胞亚群。胶原酶处理对大多数受试分子的表面表达只有轻微影响，而分散酶处理在后续的流式细胞术分析中对大多数表面标志物的抗体介导的可检测性有相当大的影响。这种影响是持久的，在高剂量分散酶处理的情况下，即使在体外培养24小时后，对大多数表面分子的影响仍然明显。值得注意的是，高剂量分散酶处理不仅影响某些分子的表面表达，还损害了CD4(+)和CD8(+) T细胞的抗原特异性增殖。总之，我们的数据表明，酶解组织对多种细胞表面分子的表达有深远影响，这对表型分析、基于流式细胞术和磁珠分选的靶细胞分离以及细胞培养实验中的免疫细胞功能都有直接影响。
Immunological characterization of immune cells that reside in specific anatomic compartments often requires their isolation from the respective tissue on the basis of enzymatic tissue disintegration. Applying enzymatic digestion of primary splenocytes, we evaluated the impact of collagenase and dispase, two enzymes that are commonly used for the liberation of immune cells from tissues, on the detectability of 48 immunologically relevant surface molecules that are frequently used for flow cytometric identification, isolation, and characterization of immune cell subsets. Whereas collagenase treatment had only minor effects on surface expression of most molecules tested, dispase treatment considerably affected antibody-mediated detectability of the majority of surface markers in subsequent FACS analyses. This effect was long lasting and, in case of high-dose dispase treatment, evident for the majority of surface molecules even after 24 h of in vitro culture. Of note, high-dose dispase treatment not only affected surface expression of certain molecules but also impaired antigen-specific proliferation of CD4(+) and CD8(+) T cells. Together, our data indicate that enzymatic tissue disintegration can have profound effects on the expression of a variety of cell-surface molecules with direct consequences for phenotypic analysis, FACS-and MACS-based target cell isolation, and immune cell function in cell culture experiments.