Detection of N-ethyl-N-nitrosourea-induced Pig-a gene mutation in rats by immunomagnetic separation technique combined with flow cytometry
10.3969/j.issn.1005-4847.2018.02.012
- VernacularTitle:免疫磁珠分离技术联合流式细胞术检测ENU致大鼠体内Pig-a基因突变
- Author:
Meirong QIN
1
;
Dandan ZHENG
;
Zeyu HUANG
;
Xiaowei WANG
;
Junpeng LI
;
Ping WANG
Author Information
1. 深圳市药品检验研究院
- Keywords:
Pig-a gene mutation;
immunomagnetic separation;
flow cytometry;
N-ethyl-N-nitrosourea;
ENU;
rats;
erythrocytes
- From:
Acta Laboratorium Animalis Scientia Sinica
2018;26(2):211-216
- CountryChina
- Language:Chinese
-
Abstract:
Objective To optimize the detection test of Pig-a gene mutation in peripheral blood of rats by enriching and detecting mutant erythrocytes, using immunomagnetic separation technique in combination with flow cytometry. Methods SD rats were administered with 20,40 and 80 mg/(kg·bw)doses of N-ethyl-N-nitrosourea(ENU) continually for 3 days. The peripheral blood samples of rats were collected on the 7th,14th and 28th days, respectively, after treatment. Immunomagnetic separation columns were used to enrich RETCD59- and RBCCD59- cells, and then flow cytometry was used to count the number of pre-column and post-column peripheral erythrocytes. Results Compared with the control group,the frequencies of RETCD59- and RBCCD59- were significantly increased in each ENU group(P<0.05). With immunomagnetic separation technique, the test of Pig-a gene mutation of a sample could be completed within 3 minutes,and the number of detected RETCD59- or RBCCD59-cells was up to 2×104or 9×104, respectively. Conclusions In this study,immunomagnetic separation in combination with flow cytometry is used to establish and optimize the Pig-a gene mutation test in rat peripheral blood,showing a high-throughput detection and improved accuracy and efficiency of the experiment.
