Fluostar optima microplate luminometer

Briefly, mice were anesthetized with isoflurane diluted in a 50% O₂ and placed in a stereotaxic frame with the head forming a nearly 135° angle with the body. Mice were kept under anesthesia during the surgery. The CSF of WT and TNAP+/- mice was collected from the cisterna magna with a pulled capillary. Blood vessels were carefully avoided when penetrating the dura mater with the capillary tube to prevent contamination from plasma proteins. CSF was placed in a tube with 100 μM ARL 67156, a competitive inhibitor of ecto-ATPases (Levesque et al., 2007 (link)) and immediately frozen on dry ice to further determine the ATP concentration.
The nucleotide concentration in the CSF was measured using ENLITEN^® rLuciferase/Luciferin reagent (Promega) according to the manufacturer’s protocol. Briefly, 1 μL CSF from WT or TNAP+/- mice was transferred to wells of a 96-well plate placed on ice. The 96-well plate was set in a FLUOstar OPTIMA Microplate Luminometer (BMG LABTECH GmbH), and 100 μL of rLuciferase/Luciferin reagent was automatically injected into each well at room temperature.

Adenosine triphosphate release was measured using the ENLITEN^® rLuciferase/Luciferin reagent (Promega). Granule cells were maintained for 30 min at 37°C in Mg²⁺-free Locke’s buffer containing 100 μM ARL67156, a competitive inhibitor of ecto-ATPases (Levesque et al., 2007 (link)), and subsequently, 50 μl of extracellular medium was collected to measure the basal ATP levels. The cells were then stimulated with 10 μM ionomycin and after 5 min, 50 μl of the extracellular medium was collected to measure ATP concentration after stimulation. The samples were centrifuged at 600 × g for 5 min at 4°C and 10 μl of supernatant were transferred onto 96-well plate placed on ice. When indicated, cells were incubated with 2 μM Evans Blue (Sigma–Aldrich) for 1 h (Loiola and Ventura, 2011 (link)). The plate was then situated in a FLUOstar OPTIMA Microplate Luminometer (BMG LABTECH GmbH) and 100 μl of rLuciferase/Luciferin reagent was automatically injected into each well at RT. The ATP concentration was estimated by interpolation from a linear standard curve.