Comparison of the LDH and MTT assays for quantifying cell death: validity for neuronal apoptosis?

Neuronal apoptosis induced in cortical cultures by exposure to serum depriv ation, staurosporine, nifedipine, or C2-ceramide was assayed by lactate deh ydrogenase (LDH) release or inhibition of 3-(4,5-dimethylthiazol-2-yl)2,5-d iphenyl bromide (MTT) reduction. The protective effects of neurotrophin-4, Z-Val-Ala-Asp-fluoromethylketone (ZVAD), and cycloheximide against each ins ult were also assayed. The level of injury for each insult was similar whet her determined by LDH release or inhibition of MTT reduction, but effects o f anti-apoptotic agents were assay dependent. ZVAD and cycloheximide protec ted 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 posit ive cells provided results consistent with values obtained using the LDH as say. These results indicate that both LDH release and MTT reduction accurat ely determine apoptotic death of neurons. However, the MTT assay does not a lways correctly quantify neuroprotective effects, this likely reflects diff erences in the point of the death pathway that the neuroprotective agents a ct. Therefore, while the MTT assay is of limited value in assessing the eff icacy of neuroprotective strategies, it may provide information regarding w hether specific anti-apoptotic agents act up or downstream of mitochondrial dysfunction. (C) 2000 Elsevier Science B.V. All rights reserved.