Department of Chemical Engineering, Northwestern University, Evanston, IL60208-3120, USA.
Biotechnol Bioeng. 1992 Oct 20;40(8):947-64. doi: 10.1002/bit.260400811.
Hybridomas are known to exhibit increased specific antibody production rated when subjected to environmental stress. Under these conditions, viability is low so that population-average measurements do not properly reflect the state of viable cells. Even for flow cytometry, which gives a population distribution, special techniques must be used to discriminate between viable and nonviable cells. We describe the use of the vital stain ethidium monoazide (EMA) for independent measurement of intracellular antibody content in live and dead cells via flow cytometry. EMA is shown to be superior to light scattering techniques in identifying dead cells. We apply this technique to show that, in control batch culture, the specific antibody production rate and antibody content in live cells are constant during exponential growth, but decrease as cells enter the stationary phase. Antibody is retained in dead cells, but at a lower level than in live cells. We further show that, under hyperosmotic stress, the specific antibody production rate and antibody content in live both remain high during death phase.
杂交瘤在受到环境压力时表现出增加的特异性抗体产生率。在这些条件下，存活率低，因此群体平均测量不能正确反映存活细胞的状态。即使对于给出群体分布的流式细胞术，也必须使用特殊技术来区分存活细胞和非存活细胞。我们描述了使用活细胞染料 ethidium monoazide (EMA) 通过流式细胞术独立测量活细胞和死细胞内的细胞内抗体含量。EMA 在识别死细胞方面优于光散射技术。我们应用该技术表明，在对照批量培养中，活细胞中的特异性抗体产生率和抗体含量在指数生长期间保持不变，但随着细胞进入静止期而下降。抗体保留在死细胞中，但水平低于活细胞。我们进一步表明，在高渗应激下，活细胞中的特异性抗体产生率和抗体含量在死亡阶段仍然很高。
