A rapid method for determination of cell survival in primary neuronal DRG cultures.
A simple and rapid enzyme-linked immunosorbent assay (ELISA) has been developed to provide an alternative to cell counting to detect increases in cell survival in primary neuronal cultures. This sensitive assay has the advantage of being less time consuming and labour intensive than cell counting, can be used to quantify cell survival and is more accurate than estimation methods of counting. The ELISA uses an antibody raised to GAP-43, a growth-associated protein which is strongly expressed by developing and regenerating neurones. The effects of nerve growth factor (NGF), neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) on GAP-43 immunoreactivity in dissociated primary cultures of rat and chick dorsal root ganglia have been compared to results obtained by cell counting. Data show that human NGF produced the greatest increase in GAP-43-immunoreactive neurones in both species; this increase in immunoreactivity correlated well with the increased survival shown by cell count data. Results prove that the ELISA can also be used to accurately detect small changes in cell survival as seen with NT-3 and BDNF, or potentiation of the effects obtained with the trophic factor NT-3. In conclusion, this ELISA may be a useful tool to detect neurotrophic effects of unknown agents or novel neurotrophins.
Young
L
L
Department of Pharmacology, Merck, Sharp and Dohme Laboratories, Neuroscience Research Centre, Harlow, Essex, UK.
Bilsland
J
J
Harper
S
S
D000818:Animals; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002642:Chick Embryo; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D019922:GAP-43 Protein / Q000378:metabolism*; D005727:Ganglia, Spinal / Q000378:metabolism*; D006801:Humans; D009414:Nerve Growth Factors / Q000378:metabolism / Q000494:pharmacology; D009474:Neurons / Q000378:metabolism*; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley
D019922:GAP-43 Protein; D009414:Nerve Growth Factors