Li Li1, Pei-dong Chi, Rui Meng, Bin-yan Yang, Chang-you Wu. 1. Department of Immunology, Zhongshan School of Basic Medicine, Sun Yat-sen University, Key Laboratory of Tropical Disease Control Research of Ministry of Education, Guangzhou 510080, China. shuicao345224@yahoo.com.cn
Abstract
AIM: To investigate the correlation of BrdU incorporation with the activation and cytokine expression of T cells. METHODS: PBMCs from healthy persons were isolated and stimulated by PMA plus Ionomycin at different periods of time, BrdU was then added to the cells one hour before the end of culture. The cells were harvested and stained with anti-BrdU, anti-cell surface and intracellular antibodies. Then the cells were washed and analyzed by flow cytometer. RESULTS: The peak of BrdU incorporation was observed in T cells after they were stimulated for 48 hours in vitro, but no further increase of the peak of BrdU incorporation was found after incubated for a longer period of time. The comparison made between BrdU incorporation and cell activation indicated CD69 expression reached the peak after stimulated for 8 hours whereas CD25 was at the peak after stimulated for 24 hours. Furthermore, no correlation between BrdU incorporation and cytokine production was observed. High frequency of IFN-gamma producing cells was detected after stimulated for 8 hours but no obvious increase was observed for a longer period of time. When PBMC were stimulated with OKT3 plus antiCD28, the percentage of BrdU(+) T cells was higher than that stimulated by PMA plus Ionomycin. Similarly, the percentage of BrdU(+) CD8(+) T cells was higher than that of BrdU(+) CD4(+) T cells. CONCLUSION: The percentage of BrdU(+) cells can be detected by flow cytometer to evaluate the proliferation of T cells. Only a few T cells proliferate after polyclonal stimulation and BrdU incorporation is dependent on stimulants and time of stimulation. Therefore, BrdU incorporation is not correlated with activation markers and cytokine production.
AIM: To investigate the correlation of BrdU incorporation with the activation and cytokine expression of T cells. METHODS: PBMCs from healthy persons were isolated and stimulated by PMA plus Ionomycin at different periods of time, BrdU was then added to the cells one hour before the end of culture. The cells were harvested and stained with anti-BrdU, anti-cell surface and intracellular antibodies. Then the cells were washed and analyzed by flow cytometer. RESULTS: The peak of BrdU incorporation was observed in T cells after they were stimulated for 48 hours in vitro, but no further increase of the peak of BrdU incorporation was found after incubated for a longer period of time. The comparison made between BrdU incorporation and cell activation indicated CD69 expression reached the peak after stimulated for 8 hours whereas CD25 was at the peak after stimulated for 24 hours. Furthermore, no correlation between BrdU incorporation and cytokine production was observed. High frequency of IFN-gamma producing cells was detected after stimulated for 8 hours but no obvious increase was observed for a longer period of time. When PBMC were stimulated with OKT3 plus antiCD28, the percentage of BrdU(+) T cells was higher than that stimulated by PMA plus Ionomycin. Similarly, the percentage of BrdU(+) CD8(+) T cells was higher than that of BrdU(+) CD4(+) T cells. CONCLUSION: The percentage of BrdU(+) cells can be detected by flow cytometer to evaluate the proliferation of T cells. Only a few T cells proliferate after polyclonal stimulation and BrdU incorporation is dependent on stimulants and time of stimulation. Therefore, BrdU incorporation is not correlated with activation markers and cytokine production.
Authors: Christiane L Schnabel; P Steinig; M Koy; H-J Schuberth; C Juhls; D Oswald; B Wittig; S Willenbrock; H Murua Escobar; C Pfarrer; B Wagner; P Jaehnig; A Moritz; K Feige; J-M V Cavalleri Journal: BMC Vet Res Date: 2015-06-23 Impact factor: 2.741