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[In vitro bromodeoxyuridine labelling of rabbit adipose-derived stromal stem cells]
Hongmian Li1, Jianhua Gao, Feng Lu
1Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou Guangdong, 510515, P.R. China.
Objective:
To explore the optimal dosage, timing and cytotoxicity of bromodeoxyuridine (BrdU) labelling for rabbit adipose-derived stromal stem cells (ADSCs) in vitro so as to confirm its feasibility for stem cells labelling and tracer means.
Methods:
Six rabbits were used in this experiment, aged 8-12 weeks, weighing 1.5-2.0 kg and neglecting their gender. 1-2 mL fat was removed, the ADSCs were isolated and cultured using the adherence method in vitro. The 3rd passage of ADSCs was incubated withBrdU at 5, 10, 15 and 20 microg/mL (groups A, B, C and D) for 12, 24, 48 and 72 hours to identify the optimal BrdU concentration and incubating time for cell labelling. Immunohistochemistry and trypanblau strain were performed respectively to calculate the labelling index (positive rate) and the cells' activity for different time after BrdU labelling. The ADSCs without BrdU labelling were used as control (Group E).
Results:
The main appearance of primary ADSCs was short fusiform shape, and of the 3rd passage ADSCs long fusiform shape. The 3rd passage of ADSCs could differentiate into osteoblasts and adipocytes under corresponding inductive medium. The ADSCs' nucleus show green fluor under fluorescence microscope after labeled by the BrdU. The labelling ratio increased in groups A, B, C and D after incubating 12 hours, the mean labelling ratio were 30.6% +/- 2.3%, 32.4% +/- 1.9%, 45.8% +/- 1.8%, 50.8% +/- 3.1%, respectively, and the labelling ratio of Group E was 0. There were significant differences between groups C, D and Group A (P < 0.01). The labelling ratio of groups A, B, C and D were 45.9% +/- 2.0%, 87.9% +/- 3.3%, 90.6% +/- 2.9%, 91.7%+/- 3.2%, respectively after 24 hours and the labelling ratio of Group E was 0. There were significant differences between groups B, C, D and Group A (P < 0.01). The results of all groups after incubating 48 hours and 72 hours were similar to that after incubating 24 hours. The cell counting of groups A, B, C and D were better than that of Group E, but showing no siginificant differences (P > 0.05).
Conclusion:
The most appropriate time for BrdU labelling ADSCs is 48 hours, the most appropriate concentration is 10 microg/mL. The labelling rate is high and cytotoxicity is little.
