During the present study, the authors explored the optimal dosage, timing and cytotoxicity for bromodeoxyuridine (BrdU) labelling of brepho-rabbit bone marrow mesenchymal stem cells (BMSCs) in vitro. This study was conducted in order to confirm its feasibility for stem cells labelling. Brepho-rabbit bone marrow was isolated and cultured using the degradient centrifugation method in vitro. The three passages of BMSCs were incubated with BrdU at different concentrations (5, 10, 15 and 20 mg/L) for incubating time (12, 24, 48 and 72 h), to identify the optimal BrdU concentration and incubating time for cell labelling. Immunohistochemical and trypan blue straining were performed respectively to calculate the labelling index (positive rate) and the cells’ activity for different time after BrdU labelling. The observation of bmSCs’ culture showed the main appearance of primary BMSCs was wide, applanate and short fusiform shape, and long fusiform shape was the main appearance of passage 3 BMSCs. The passage 3 of BMSCs could be differentiated into osteoblast and adipocyte under corresponding inductive medium. The observation of immunohistochemical suggested the BMSCs’ nucleus showed green flour under fluorescence microscope after labelled by the BrdU. The labelling ratio increasingly elevated of Groups A, B, C and D after the BMSCs were incubated 12 h, their mean labelling ratio was (33.2(4.3)%, (34.5(3.9)%, (47.9(4.8)% and (52.8(5.1)%, respectively, and the labelling ratio of group E is zero, which were significantly difference among the groups A, B and D (P<0.01).The labelling ratio of groups A, B, C and D was (48.1(4.0)%, (83.4(5.3)%,(91.7(4.9)% and (92.6(4.2)%, respectively after the BMSCs were incubated 24 h, and the labelling ratio of group E is zero. The results of all groups after incubating 48 h, 72 h were similar to those of 24 hour. The rate of label increased gradually with time and concentration, more than 90% BMSCs were labelled after 48 h and when the concentration of BrdU beyond 10 mg/L, at the same time, the living cell more than 90%. The optimal time for labelling BMSCs is 48 h, the most appropriate concentration is 10 mg/L. The labelling rate of BrdU is very high and its cytotoxicity is very small, therefore it is safe.
Authors: Li, Dehui; Li, Hongmian; Liang, Ziqian; Liu, Dalie ;Full Source: Guangxi Yike Daxue Xuebao 2011, 28(4), 506-511 (Ch) ;