Assessment of physiologic natural killer cell cytotoxicity in vitro
Assessment of physiologic natural killer cell cytotoxicity in vitro
复制标题
DOI：
10.1016/j.humimm.2011.08.006
复制
发表时间：
2011-11-01
期刊：
影响因子：
2.7
通讯作者：
Eiermann, Thomas
中科院分区：
文献类型：
--
作者：
Duske, Heidi;Sputtek, Andreas;Eiermann, Thomas
在这里，我们描述了一种改进的 (51) 铬释放测定 (CRA)，以比较供体自然杀伤 (NK) 细胞的活性。为了验证该测定，我们分析了 NK 细胞的样品制备、孵育和冷冻保存。根据 NK 细胞的百分比校正效应器与靶标的比率。将对数曲线拟合到 CRA 的数据以计算最大活性。将特异性裂解标准化为参考样品，并标准化为参考的平均特异性裂解。我们发现，添加和去除 DMSO 所涉及的时间跨度较长，可以增加 NK 细胞活性的恢复。冷冻和解冻降低了 NK 细胞的细胞毒性，但维持了新鲜制备的 NK 细胞之间的相对差异。相反，解冻细胞的培养基孵育显着增加了细胞毒性潜力，但也扰乱了这些相对差异。当用 IL-2 刺激细胞时，这些结果基本相等。总之，我们建立了一种使用冷冻保存的外周血单核细胞的标准化测定方法，作为研究个体生理 NK 细胞活性的适当工具。该测定可能有助于预测供体 NK 细胞的体内活性，以协调移植研究中获得的 NK 细胞的相互矛盾的数据。 (C) 2011 年美国组织相容性和免疫遗传学学会。由爱思唯尔公司出版。保留所有权利。
Here, we describe an improved (51)chromium release assay (CRA) to compare donor natural killer (NK) cell activity. To validate the assay, we analyzed sample preparation, incubation, and cryopreservation of NK cells. The effector-to-target ratio was corrected for the percentage of NK cells. A logarithmic curve was fitted to the data of the CRA for calculation of the maximum activity. The specific lysis was standardized to a reference sample and normalized to the mean specific lysis of the reference. We found that a longer time span involved with both the addition and the removal of DMSO increased the recovery of NK cell activity. Freezing and thawing reduced the cytotoxicity of NK cells but sustained the relative differences that were seen between freshly prepared NK cells. In contrast, medium incubation of thawed cells markedly increased the cytotoxic potential but also deranged these relative differences. Those were widely equalized when cells were stimulated with IL-2. In conclusion, we established a standardized assay with cryopreserved peripheral blood mononuclear cells as an appropriate tool for investigation of individual physiologic NK cell activity. This assay may help to predict donor NK cell activity in vivo, to reconcile conflicting data about NK cells obtained in transplantation studies. (C) 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.