Quantification of neuron survival in monolayer cultures using an enzyme-linked immunosorbent assay approach, rather than by cell counting.
The determination of neurotoxicity in monolayer mixed cultures has traditionally necessitated the time consuming and subjective procedure of counting neurons. In this paper, we propose a modification of an immunohistochemical staining method with a neuron-specific antibody against MAP2, that allows for quantification of neuron number to be done using an enzyme-linked immunosorbent assay (ELISA) plate reader. This new procedure involves the use of the compound 2,3'-azino-bis(ethylbenzothiazoline-6-sulphonic acid) (ABTS) at the last stage of the staining procedure. We employed two neurotoxicity models (the excitotoxin kainic acid and the interactions between gp120, the glycoprotein of HIV, and the stress hormone corticosterone) to compare the results obtained with this new method and the old method of immunohistochemical staining followed by 3,3'-daminobenzidine (DAB) and the counting of neurons. The ABTS/ELISA method was found to be a fast, reliable and objective procedure for the quantification of neurotoxicity.
Brooke
S M
SM
Department of Biological Sciences, Stanford University, CA 94305, USA. sheila.brooke@stanford.edu
Bliss
T M
TM
Franklin
L R
LR
Sapolsky
R M
RM
D001253:Astrocytes / Q000737:chemistry / Q000166:cytology; D002452:Cell Count / Q000379:methods; D018929:Cell Culture Techniques / Q000379:methods; D002470:Cell Survival; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006624:Hippocampus / Q000737:chemistry / Q000166:cytology; D006801:Humans; D007150:Immunohistochemistry; D009474:Neurons / Q000737:chemistry / Q000166:cytology*