Determination of intracellular ATP in primary cultures of neurons.
The aim of this work was to develop and characterize a quick and simple procedure to determine the intracellular content of ATP in monolayer primary cultures of neurons. The baseline was to use the minimum amount of cells which still provides reproducible results. The first step consists of releasing intracellular ATP from the cells. This is accomplished by treatment with a detergent solution, the somatic cell releasing reagent from Sigma. This reagent is claimed by the manufacturer to release ATP from a suspension of viable somatic cells. The procedure has been adapted to be used for attached cells (neurons or astrocytes growing in monolayer), thus avoiding the use of alternative time-consuming procedures to release ATP such as boiling buffers or trichloroacetic acid. After its release the free ATP was measured using the firefly luciferase reaction. We have used this protocol to assess the effect of neurotoxic concentrations of glutamate on the intracellular content of ATP in neurons. The same procedure has been used successfully to determine intracellular ATP in primary cultures of astrocytes.
Marcaida
G
G
Instituto de Investigaciones Citológicas, Fundación Valenciana de Investigaciones Biomédicas, Valencia, Spain.
Miñana
M D
MD
Grisolía
S
S
Felipo
V
V
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D003902:Detergents / Q000494:pharmacology; D018698:Glutamic Acid / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism*; D008156:Luciferases; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009488:Neurosciences / Q000379:methods*; D009498:Neurotoxins / Q000494:pharmacology; D009994:Osmolar Concentration; D051381:Rats; D017208:Rats, Wistar
D003902:Detergents; D009498:Neurotoxins; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D008156:Luciferases