A sensitive and selective assay of neuronal degeneration in cell culture.
We have developed a simple and sensitive assay to quantify neuron-specific death in primary cell cultures that represents a significant improvement over more commonly used methods including manual cell counting and lactate dehydrogenase release. This new method selectively detects neuronal death by combining immunolabeling for a neuron-specific marker with the ease, sensitivity, and speed of an enzyme-linked fluorescence assay. Using microtubule associated protein 2 (MAP2) as a neuron-specific marker, we assessed glutamate-receptor mediated neurotoxicity in neuron-enriched cultures and in mixed neuronal/glial cultures established from mouse forebrain and compared these results to neuronal death measured by lactate dehydrogenase (LDH) release. We were able to achieve statistically significant differences in toxicity between intermediately toxic concentrations of glutamate (30, 50, and 100 microM) with the MAP2 assay, while we were not able to discriminate among these concentrations with the LDH assay. We were also able to measure hydrogen peroxide-induced neuronal death, and demonstrate neuroprotection by antioxidant addition. This new assay is easily adaptable to high-throughput in vitro screens of neurodegeneration and of neuroprotective therapies.
Division of Pharmacology, The Ohio State University, Columbus, 43210, USA.
Raeann L
RL
Carrier
Thong C
TC
Ma
Karl
K
Obrietan
Kari R
KR
Hoyt
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001681:Biological Assay; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques; D018846:Excitatory Amino Acids / Q000633:toxicity; D005455:Fluorescent Antibody Technique; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D018698:Glutamic Acid / Q000633:toxicity; D006861:Hydrogen Peroxide / Q000633:toxicity; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009410:Nerve Degeneration / Q000139:chemically induced / Q000201:enzymology / Q000473:pathology*; D009457:Neuroglia / Q000502:physiology; D018696:Neuroprotective Agents / Q000494:pharmacology; D010078:Oxazines; D016877:Oxidants / Q000633:toxicity
D000975:Antioxidants; D018846:Excitatory Amino Acids; D005904:Glial Fibrillary Acidic Protein; D008869:Microtubule-Associated Proteins; D018696:Neuroprotective Agents; D010078:Oxazines; D016877:Oxidants; D018698:Glutamic Acid; C014180:resorufin; D006861:Hydrogen Peroxide; D007770:L-Lactate Dehydrogenase