Detection of N-ethyl-N-nitrosourea-induced Pig-a gene mutation in rats by immunomagnetic separation technique combined with flow cytometry
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(Shenzhen Institute for Drug Control, Shenzhen Key Laboratory of Drug Quality Standard Research, Shenzhen 518057, China)

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Shenzhen Science and Technology Planning Project (No. JCYJ20140418153354413)

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    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 RET CD59 - and RBC CD59- 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 RET CD59- and RBC CD59- 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 RET CD59 - or RBC CD59 - cells was up to 2 ×10 4 or 9 ×10 4 , 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.

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History
  • Received:December 02,2017
  • Revised:
  • Adopted:
  • Online: May 14,2018
  • Published: