연구논문 : 피부감작능 평가를 위한 BrdU-FCM 국소림프절시험법 기법 이전을 위한 제 조건

Local lymph node assay (LLNA) using mice has been adopted as an alternative model for testing skin sensitization to chemicals. Modification of LLNA was recently reported through combination of BrdU incorporation and flow cytometric analysis, which was designated as LLNA:BrdU-FCM. We introduce how LLNA:BrdU-FCM could be successfully established in a naive laboratory where the method was never experienced. Nine week old female BALB/c mice, 5 mice per group, were used and grouped into non-treatment, BrdU only-treatment, acetone-olive oil (AOO) vehicle-treatment, and 5, 10, 25% α-hexylcinamaldehyde (HCA) positive control. BrdU solution was injected intraperitoneally one day after three consecutive application of AOO or HCA on dorsum area of both ears. Auricular lymph node was collected from both ears one day after the BrdU injection. Thereafter flow cytometric staining procedures were followed. Various parameters related with calculation of stimulation index were analyzed including number of lymphocytes, BrdU incorporation%, and number of BrdU incorporated lymphocytes. The critical procedures for successful establishment were as followings; quality control of every quantitative apparatus, appropriate application of test compounds on dorsum of mouse ear, precise injection of BrdU, accurate preparation of single cell suspension and counting of lymph node cells, and experienced operation of flow cytometer. Face-to-face training from an experienced lead laboratory and preparation of very detailed standard operational procedures could play the most important role for the establishment. Based on Performance Standards for Assessment of Proposed Similar or Modified Test Methods by OECD, the LLNA:BrdU-FCM could be a good alternative method for screening skin sensitizers.