Optimized dissociation protocol for isolating human glioma stem cells from tumorspheres via fluorescence-activated cell sorting
Optimized dissociation protocol for isolating human glioma stem cells from tumorspheres via fluorescence-activated cell sorting
复制标题
通过荧光激活细胞分选从肿瘤球中分离人神经胶质瘤干细胞的优化解离方案
DOI：
10.1016/j.canlet.2016.04.022
复制
发表时间：
2016-07-10
期刊：
影响因子：
9.7
通讯作者：
Bian, Xiu-wu
中科院分区：
文献类型：
--
作者：
Lv, Donglai;Ma, Qing-hua;Bian, Xiu-wu
基于表面标记CD133的荧光激活细胞分选（FACS）是从异质胶质瘤细胞群中分离胶质瘤干细胞（GSCs）的最常用方法。该方法的优化将对未来的GSC研究产生深远的影响。使用Liberase-TL、trypsin、TrypLE、Accutase和非酶解细胞解离液（NECDS）这5种常用的消化试剂，分离来源于2个原发胶质瘤标本（091214和090116）和细胞系U87和T98G的胶质瘤肿瘤球。评估解离时间、细胞活力、CD133的保留率和干细胞容量。结果表明，Liberase-TL （200 μ g/ml）组单细胞活力高，对CD133抗原损伤小。然而，NECDS将肿瘤球分离成单个细胞的效率相当低。同时，该消化试剂的使用导致了明显的细胞和抗原损伤。综上所述，Liberase-TL （200 μ g/ml）是从肿瘤球中分离GSCs的理想试剂。相反，应该仔细考虑将NECDS用于这种协议。2016爱思唯尔爱尔兰有限公司版权所有。
Fluorescence-activated cell sorting (FACS) based on the surface marker CD133 is the most common method for isolating glioma stem cells (GSCs) from heterogeneous glioma cell populations. Optimization of this method will have profound implications for the future of GSC research. Five commonly used digestion reagents, Liberase-TL, trypsin, TrypLE, Accutase, and non-enzymatic cell dissociation solution (NECDS), were used to dissociate glioma tumorspheres derived from two primary glioma specimens (091214 and 090116) and the cell lines U87 and T98G. The dissociation time, cell viability, retention of CD133, and stemness capacity were assessed. The results showed that single cells derived from the Liberase-TL (200 mu g/ml) group exhibited high viability and less damage to the antigen CD133. However, the efficiency of NECDS for dissociating the tumorspheres into single cells was fairly low. Meanwhile, the use of this digestion reagent resulted in obvious cellular and antigenic impairments. Taken together, Liberase-TL (200 mu g/ml) is an ideal reagent for isolating GSCs from tumorspheres. In contrast, the use of NECDS for such a protocol should be carefully considered. (C) 2016 Elsevier Ireland Ltd. All rights reserved.