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 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-tetrazolium 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.
Lobner
D
D
Room 426, Department of Biomedical Sciences, Marquette University, 561 N. 15th Street, Milwaukee, WI 53233, USA. lobnerd@vms.csd.mu.edu
D000818:Animals; D017209:Apoptosis; D002452:Cell Count / Q000379:methods; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D004396:Coloring Agents; D003513:Cycloheximide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005333:Fetus; D007770:L-Lactate Dehydrogenase / Q000032:analysis*; D051379:Mice; D009414:Nerve Growth Factors / Q000494:pharmacology; D009457:Neuroglia / Q000166:cytology; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D018696:Neuroprotective Agents / Q000494:pharmacology; D015203:Reproducibility of Results; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D013778:Tetrazolium Salts; D013844:Thiazoles; D014343:Trypan Blue
D004396:Coloring Agents; D004791:Enzyme Inhibitors; C064769:N-acetylsphingosine; D009414:Nerve Growth Factors; D018696:Neuroprotective Agents; D013778:Tetrazolium Salts; D013844:Thiazoles; D003513:Cycloheximide; D007770:L-Lactate Dehydrogenase; C022616:thiazolyl blue; D014343:Trypan Blue; D013110:Sphingosine; C073922:neurotrophin 4