Autengruber A, Gereke M, Hansen G, Hennig C, Bruder D
Eur J Microbiol Immunol (Bp). 2012 Jun;2(2):112-20. doi: 10.1556/EuJMI.2.2012.2.3. Epub 2012 Jun 13.
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.
对特定解剖区域内的免疫细胞进行免疫特征分析通常需要基于酶促组织分解从相应组织中分离出这些细胞。通过对原代脾细胞进行酶消化，我们评估了胶原酶和 dispase（常用于从组织中释放免疫细胞的两种酶）对 48 种常用于流式细胞术鉴定、分离和表征免疫细胞亚群的免疫相关表面分子检测能力的影响。虽然胶原酶处理对大多数测试分子的表面表达影响较小，但 dispase 处理在随后的流式细胞术分析中显著影响了大多数表面标志物的抗体介导检测能力。这种影响持续时间长，在高剂量 dispase 处理的情况下，即使在体外培养 24 小时后，大多数表面分子的这种影响仍然明显。值得注意的是，高剂量 dispase 处理不仅影响某些分子的表面表达，还损害 CD4(+)和 CD8(+)T 细胞的抗原特异性增殖。总之，我们的数据表明，酶促组织分解可对多种细胞表面分子的表达产生深远影响，这对细胞表型分析、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具有直接影响。
