Comparison of the LDH and MTT assays for quantifying cell death: validity for neuronal apoptosis?
Comparison of the LDH and MTT assays for quantifying cell death: validity for neuronal apoptosis?
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DOI：
10.1016/s0165-0270(99)00193-4
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发表时间：
2000-03-15
影响因子：
3
通讯作者：
Lobner, D
中科院分区：
文献类型：
--
作者：
Lobner, D
通过乳酸脱氢酶(LDH)释放或抑制3-(4，5-二甲基噻唑-2-基)-2，5-二苯基溴(MTT)还原来检测血清剥夺、星形孢子素、硝苯地平或C2-神经酰胺诱导的皮质细胞凋亡。同时检测神经营养素-4、Z-Val-Ala-Asp-氟甲基酮(ZVAD)和放线菌酮对各种损伤的保护作用。无论是通过LDH释放还是通过抑制四甲基偶氮唑蓝还原来确定，每种损伤的程度都是相似的，但抗凋亡药物的效果是依赖于检测的。用乳酸脱氢酶释放法检测ZVAD和放线菌酮对硝苯地平诱导的神经元死亡有保护作用，但不能用四甲基偶氮唑蓝还原试验检测。相反，只有放线菌酮能减弱C2-神经酰胺诱导的LDH释放，而zVAD和放线菌素实际上增强了C2-神经酰胺对细胞内MTT值的抑制。台盼蓝阳性细胞计数结果与乳酸脱氢酶检测结果一致。这些结果表明，乳酸脱氢酶释放和四甲基偶氮唑蓝还原都能准确地判断神经元的凋亡性死亡。然而，四甲基偶氮唑盐试验并不总是正确地量化神经保护作用，这可能反映了神经保护剂作用的死亡途径的点的差异。因此，虽然MTT法在评估神经保护策略的有效性方面的价值有限，但它可能提供关于特定的抗凋亡药物是否在线粒体功能障碍的上游或下游起作用的信息。(C)2000 Elsevier Science B.V.保留所有权利。
Neuronal apoptosis induced in cortical cultures by exposure to serum deprivation, staurosporine, nifedipine, or C2-ceramide was assayed by lactate dehydrogenase (LDH) release or inhibition of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl bromide (MTT) reduction. The protective effects of neurotrophin-4, Z-Val-Ala-Asp-fluoromethylketone (ZVAD), and cycloheximide against each insult were also assayed. The level of injury for each insult was similar whether determined by LDH release or inhibition of MTT reduction, but effects of anti-apoptotic agents were assay dependent. ZVAD and cycloheximide protected neurons from nifedipine-induced death, when assayed by LDH release, but not MTT reduction. In contrast, only cycloheximide attenuated C2-ceramide-induced LDH release, while ZVAD and cycloheximide actually enhanced the C2-ceramide induced inhibition of MTT reduction. Counting of trypan blue positive cells provided results consistent with values obtained using the LDH assay. These results indicate that both LDH release and MTT reduction accurately determine apoptotic death of neurons. However, the MTT assay does not always correctly quantify neuroprotective effects, this likely reflects differences in the point of the death pathway that the neuroprotective agents act. Therefore, while the MTT assay is of limited value in assessing the efficacy of neuroprotective strategies, it may provide information regarding whether specific anti-apoptotic agents act up or downstream of mitochondrial dysfunction. (C) 2000 Elsevier Science B.V. All rights reserved.