摘要
目的制备稳定可用的流式细胞仪校准用鸡红细胞。方法引入明胶沉降、无Ca2+Hank's平衡盐缓冲液处理及低速离心等方法对传统鸡红细胞分离方法进行改良;并采用Triton X-100打孔及Rnase酶降解RNA的方法增强细胞DNA染色效果,检测所得新鲜细胞活率及固定后细胞DNA含量在流式细胞仪上的变易系数(CV)值,并与传统方法获得的鸡红细胞进行对比。结果改良后方法获得的鸡红细胞活率(98.5±3.5)%高于传统方法(93.5±2.7)%(P<0.05)。经戊二醛固定后,该细胞在90 d的保存时间内基本稳定,且其CV值显著低于传统方法([6.0±0.3)%-(6.2±0.4)%vs(8.6±0.5)%-(13.1±1.4)%,P<0.01)]。结论改良后方法获得的鸡红细胞可被开发作为流式细胞仪校准的有效工具。
Objective To prepare stable chicken red blood cells for the calibration of flow cytometry. Methods The traditional isolation method of chicken red blood cells was modified by incorporating gelatin technique, Ca^2＋-free HBSS treatment and low-speed centrifugation. The effect of fluroscence staining of the cells was improved by the addition of TritonX-100 to enhance the membrane permeability and Rnase enzymes to disintegrate RNA tiles. The modified method was compared with the traditional method for viability of the freshly isolated cells and the DNA content coefficient of variation （CV） of the fixed cells. Results Chicken red blood cells obtained by the modified method showed a significantly higher viability than those obtained by the traditional method [（98.5±3.5）% vs （93.5±2.7）%, P〈0.05]. After glutaraldehyde fixation, the isolated cells with the modified method were stable during the 90-day preservation with a significantly lower CV than the cells obtained by the traditional method [（6.0±0.3）% to 6.2±0.4% vs （8.6±0.5）% to （13.1 ± 1.4）%, P〈0.01]. Conclusion The chicken red blood ceils isolated using the modified method can be applicable for calibration of flow cytometry.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2013年第1期57-60,共4页
Journal of Southern Medical University
基金
863项目(2011AA02A106)
中国科学院知识创新工程重要方向项目(KGCX2-YW-912)
江苏省产学研前瞻性联合研究项目(BY2011185)~~
关键词
流式细胞术
鸡红细胞
荧光
变异系数
稳定性
flow cytometry
chicken red blood ceils
fluorescence
coefficient of variation
stability
