Astrocyte Responses to Ethanol and Choline

Astrocytes plated on glass coverslips placed in 24-well plates were incubated with 1 mL DMEM/0.1% BSA medium containing 25, 50, or 75 mM ethanol or control (ethanol-free) medium for 24 h. Ethanol treatments took place in sealed chambers with a dish of water containing the same alcohol concentration present in the cultures; each chamber contained astrocytes treated with a single ethanol concentration.^{25 (link)} A gas mixture of 5% CO₂/95% air was run through these chambers, after which the chambers were sealed and incubated at 37°C for 24 h. choline treatments were carried out by incubating astrocyte cultures with 1 mL DMEM/0.1% BSA medium containing 0 (control), 10, 50, or 100 mM choline chloride (Sigma Aldrich, St. Louis, MO) for 24 h. To test the effects of both ethanol and choline on neurite outgrowth, astrocytes were incubated with 75 mM ethanol, 100 mM choline, 75 mM ethanol + 100 mM choline, or control (treatment-free) DMEM/0.1 BSA medium for 24 h.