DETECTION OF B LYMPHOMA CELLS UNDERGOING APOPTOSIS BY ANNEXIN-V ASSAY

来源 :Chinese Medical Sciences Journal | 被引量 : 0次 | 上传用户:manaijin
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To quantitatively analyze apoptotic and secondary necrotic cells unde r apoptosis conditions. Methods. The cells of Burkitt lymphoma (BL) cell line Raji were incubated with 1 .0 μmol/L dexamethasone (DEX) for 2, 4 and 8 h respectively, then stained with Annexin V FITC (fluorescein isothiocyanate conjugated) which was used to detec t the exposed phosphatidylserine (PS) on the epimembrane resulting from a loss o f phospholipid asymmetry in the early stage of apoptosis, and also stained with propidium iodide (PI) which allows analysis of secondary necrotic cells related with cell membrane and DNA damage that probably represent late stage of apoptosi s, then apoptotic cells were quantified by flow cytometry (FCM). Furthermore, An nexin+/PI and Annexin+/PI+ cells were sorted by fluoresence activated cell sorter (FACS), and identified by electron microscopy (EM) and DNA gel electroph oresis. Results. The percentage of apoptotic cells was found to increase with the incuba tion time (r=0.97). This method was sensitive with low detection limit (0.02%), and was reproducible with low coefficient variance (CV)(4.2%). Meanwhile, the Annexin+/PI and Annexin+/PI+ cells were identified as apoptotic and necroti c cells under EM, and DNA extracted from the Annexin+/PI cells was characteri zed by “ladder pattern”. Conclusions. Annexin V assay is a specific, sensitive, accurate, reproductive and quantitative method for analyzing apoptotic cells. To quantitatively analyze apoptotic and secondary necrotic cells unde r apoptosis conditions. Methods The cells of Burkitt lymphoma (BL) cell line Raji were incubated with 1.0 μmol / L dexamethasone (DEX) for 2, 4 and 8 h respectively, then stained with Annexin V FITC (fluorescein isothiocyanate conjugated) which was used to detec t the exposed phosphatidylserine (PS) on the epimembrane resulting from a loss of phospholipid asymmetry in the early stage of apoptosis, and also stained with propidium iodide (PI) which allows analysis of secondary necrotic cells related with cell membrane and DNA damage that probably represent late stage of apoptosi s, apoptotic cells were quantified by flow cytometry (FCM). Furthermore, An nexin + / PI and Annexin + / PI + cells were sorted by fluoresence activated cell sorter (FACS), and identified by electron microscopy (EM) and DNA gel electrophoresis. Results. The percentage of apoptotic cells was found to increase with the incubation time (r = 0.97). This method was sensitive with low detection limit (0.02%), and was reproducible with low coefficient of variance (CV) (4.2%). Meanwhile, the Annexin + / PI and Annexin + / PI + necrotc c cells under EM, and DNA extracted from the Annexin + / PI cells was characteri zed by “ladder pattern”. Conclusions. Annexin V assay is a specific, sensitive, accurate, reproductive and quantitative method for apoptotic cells.
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