Ishaque A, Al-Rubeai M
Animal Cell Technology Group School of Chemical Engineering, The University of Birmingham, UK.
J Immunol Methods. 1998 Dec 1;221(1-2):43-57. doi: 10.1016/s0022-1759(98)00166-5.
Accurate identification and quantitation of apoptosis is essential for developing efficient strategies for optimisation of culture viability and productivity in cell lines of industrial significance. We have examined the possibility of using carboxy-seminaphthorhodafluor-1-acetoxymethylester (carboxy SNARF-1-AM), a pH sensitive fluoroprobe and FITC-labelled annexin V (AV), a probe specific to phosphatidylserine exposed on the surface of apoptotic cells, to monitor apoptosis and to determine the relationship between intracellular pH (pHi), apoptosis and cell cycle in hybridoma cells. Temporal changes in the distribution of proliferative capacity (S phase), metabolic activity (pHi), and cell death population dynamics were effectively and reliably determined using flow cytometry. Intracellular acidification was shown to precede the occurrence of apoptosis during batch culture and after treatment with campothecin, staurosporine and under adverse bioreactor conditions such as glutamine deprivation and oxygen deficiency. These results showed that the decrease in pHi can be used as an indicator of cellular deterioration and cell death. AV in combination with propidium iodide permitted the identification of viable, transient apoptotic and necrotic cells in heterogeneous cultures of control (PEF) cells. Hybridoma cells over-expressing bcl-2 were protected from intracellular acidification and phosphatidylserine exposure, which was associated with the suppression of apoptosis in these cells. A decrease in pHi was apparent even before the accumulation of the normally acidic G1 phase and the development of a sub-G1 region, characteristic of apoptotic cell behaviour. The pHi assay can therefore be used as a tool to predict future cell culture performance. reserved.
准确识别和定量细胞凋亡对于制定优化具有工业意义的细胞系培养活力和生产力的有效策略至关重要。我们研究了使用羧基-半萘罗丹明氟-1-乙酰氧基甲酯（羧基SNARF-1-AM，一种pH敏感荧光探针）和异硫氰酸荧光素标记的膜联蛋白V（AV，一种对凋亡细胞表面暴露的磷脂酰丝氨酸具有特异性的探针）来监测细胞凋亡，并确定杂交瘤细胞中细胞内pH值（pHi）、细胞凋亡和细胞周期之间关系的可能性。使用流式细胞术有效地、可靠地测定了增殖能力（S期）、代谢活性（pHi）和细胞死亡群体动态分布的时间变化。在分批培养期间以及用喜树碱、星形孢菌素处理后，以及在谷氨酰胺剥夺和缺氧等不利生物反应器条件下，细胞内酸化被证明先于细胞凋亡的发生。这些结果表明，pHi的降低可作为细胞恶化和细胞死亡的指标。AV与碘化丙啶结合可在对照（PEF）细胞的异质培养物中鉴定存活、短暂凋亡和坏死细胞。过表达bcl-2的杂交瘤细胞受到保护，免受细胞内酸化和磷脂酰丝氨酸暴露，这与这些细胞中细胞凋亡的抑制有关。甚至在正常酸性G1期积累和亚G1区域出现之前，pHi就明显降低，这是凋亡细胞行为的特征。因此，pHi测定可作为预测未来细胞培养性能的工具。保留。
