Primary cortical neurons were prepared from C57BL/6 mouse brains according to the established protocol [10 (link)]. Briefly, cortices were isolated from P0 pups and digested with 100 U of papain for 30 min at 37 °C and mechanically disrupted by pipetting several times in neurobasal medium (Invitrogen, Carlsbad, CA, USA) supplemented with 2% B27 (Invitrogen) and a 1% glutamine/glutamax mixture (1:3) (Invitrogen). The dissociated cortical neurons were seeded in poly-D-lysine-coated 24-well plates (0.5 × 104 cells/well) or 8-well glass slide chambers (2 × 103 cells/well) for cAMP or neurite outgrowth analysis, respectively.
Neurons were stained for β-III tubulin (1:200, cat# 5568, Cell signaling, Danvers, MA, USA) and images were acquired using a Zeiss LSM 700 confocal laser-scanning microscope. The neurite length was measured using NIH Image J software (National Institutes of Health, Bethesda, MD, USA) as we described earlier [10 (link)].
Neurons were stained for β-III tubulin (1:200, cat# 5568, Cell signaling, Danvers, MA, USA) and images were acquired using a Zeiss LSM 700 confocal laser-scanning microscope. The neurite length was measured using NIH Image J software (National Institutes of Health, Bethesda, MD, USA) as we described earlier [10 (link)].
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