流式细胞仪筛选环磷酰胺诱导骨髓嗜多染红细胞微核的技术方法

目的利用单一荧光试剂吖啶橙(acridine orange,AO)和简单的单激光流式细胞仪(FCM)计数骨髓嗜多染红细胞的微核率,达到了解化合物诱导微核作用的目的。方法分别以环磷酰胺(CP)处理雄性KM小鼠和SD大鼠,采用AO荧光染色和单激光流式细胞仪检测小鼠和大鼠骨髓含微核的嗜多染红细胞(MNPCE)及含微核的成熟红细胞(MNNCE)的微核率,并对结果进行比较分析。结果随着CP浓度的增高,骨髓中MNPCE和MNNCE也相应增多,呈良好的量效关系。实验结果同时和手工计数比较,没有显著差异。结论 AO_FCM自动化检测方法完全适用于小鼠和大鼠骨髓MNPCE及MNNCE微核率的检测。

Technical methods on screening cyclophosphamide induced bone marrow micronucleus polychromatic erythrocytes by flow cytometer

Objective To discriminate whether chemical compounds are micronuclei-inducing by counting the ratio of bone marrow micronucleus polychromatic erythrocytes (MNPCE) dyed with a single fluorescence reagent (acriding organe, AO) by single-laser flow cytometry. Methods Treating male KM mice and SD rat with cyclophosphamide (CP) respectively, and counting their frequencies of micronucleated polychromatic erythrocytes (MNPCE) as well as frequencies of micronucleated normochromatic erythrocytes (MNNCE) in bone marrow by AO and a single_laser flow cytometer (FCM), comparing and analyzing the results from different methods. Results The results showed that, along with increasing dose of CP, the ratio of MNPCE also corresponding increase, suggesting significant quantative-efficiency correlation. MNPCE were also counted manually by fluorescence microscopy, and the results showed no significant difference with that by flow cytometry. Conclusions AO_FCM fully automated detection method for the detection of MNPCE and MNNCE in mice and rat bone marrow micronucleus rate is reliable.