After 2-h adhesion, the plating medium was carefully aspirated and myelination medium was slowly added into the wells (700 μl each well for a 12 well plate). The cover slips were firmly pushed down to the bottom of culture wells with a pipette tip. We initially tried either N2 or NBM (with B27 supplement) as the myelination medium, but only limited amount of myelination was observed. However, with Inhibitors,research,lifescience,medical the combination of N2 and NBM (1:1) yielded robust myelination in the spinal cord derived culture. For the first week of culture, NGF (50 ng/mL)
and NT-3 (10 ng/mL) were included in the medium. The medium was changed every three days by replacing two-third of the medium with fresh medium. The day of the primary culture is defined as day 1 in vitro (DIV1). At DIV10, insulin was excluded from N2 and the ratio of the insulin-free N2 to NBM was Cytoskeletal Signaling inhibitor adjusted to 4:1 to prevent cell overgrowth. The final concentrations of Inhibitors,research,lifescience,medical individual component in N2 medium (DMEM-F12 based, high glucose, Invitrogen) are listed as following: insulin (10 μg/mL), transferrin (50 μg/mL), sodium selenite (5.2 ng/mL), hydrocortisone (18 ng/mL), putrescine (16 μg/mL), progesterone (6.3 ng/mL), biotin (10 ng/mL), N-acetyl-L-cysteine Inhibitors,research,lifescience,medical (5 μg/mL), BSA (0.1%),
and penicillin–streptomycin (50 units/mL). The procedures for cortex-derived culture are rather similar to those Inhibitors,research,lifescience,medical described from the spinal cord. After removing the meninges and other connective tissue, the entire cerebral cortex from both hemispheres was dissected out and pooled together from six embryos. Typically, total number of dissociated cells from the cortex is much higher (~10-fold) than from the spinal cord. Under such preparation, T3 was introduced Inhibitors,research,lifescience,medical to the
myelination medium at DIV10. Immunocytochemistry The cultured cells were rinsed with ice-cold PBS and fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature (RT). Following washing in PBS, cells were permeabilized with 0.5% Triton X-100 for 20 min, and blocked with a solution containing 10% normal goat serum/1% BSA and 0.1% Triton X-100 for 1 h. Cells were then incubated in the primary antibodies diluted in PBS/10% serum overnight. After washing, cells were incubated with biotin- or fluorescein-labeled second antibodies (mouse or rabbit IgG conjugated with Alex 488/555) ADP ribosylation factor for 1 h at RT, followed by incubation with avidin fluorescein (Alex 488 or 555) in PBS for 30 min. Cover slips were then washed and air dried, and viewed under a fluorescence microscope (Oly-750 from Olympus, Pittsburgh, PA, USA) with proper filters. For immunostaining of O4, primary antibody was applied before fixation. DAPI (1.5 μg/mL) was used in the mounting medium to counterstain the nuclei. Images were captured with a CCD camera, and superimposed using the Adobe Photoshop (version 7.