Glioma Culture
NG97 cells were grown in plastic flasks (25 cm2) with RPMI 1640 medium (Sigma Chemical Co., St Louis, MO), supplemented with 50 μM 2-ME, 2 mM L- glutamine, 100 μg/mL garamycin and 20% inactivated fetal bovine serum (complete medium). The cultures were incubated at 37°C in an atmosphere containing 95% air and 5% CO2. The medium was changed after intervals of 48 hs and when the culture reached confluence, the subculture was performed by treatment with 0.05% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA).
Immunocytochemistry
Immunocytochemical analysis of glial markers (GFAP, vimentin and S-100 protein) was performed by using specific antibodies purchased from Dako Envision+ Systems/HRP (Dako Corporation, Carpinteria, CA). Briefly, cultured NG97 cells were harvested (at passage 15), washed using low speed centrifugation (150 × g, 10 minutes) and ressuspended in complete medium. Then, cells were cyto-centrifugated on glass slides, dried at room temperature for 15 minutes and fixed in cold acetone for 15 minutes at -20°C. After a thorough wash with 0.5% BSA in PBS, the cells were treated with polyclonal rabbit anti-GFAP, monoclonal anti-vimentin and polyclonal rabbit anti-S100 antibodies according to the manufactures' instructions. The bound primary antibody was detected using peroxidase labeled polymer conjugated to either mouse or rabbit secondary antibodies. Subsequently, the slides were incubated with a substrate mixture of 3,3-diaminobenzidine (DAB) and 0.02% H2O2. Cells were then counterstained with haematoxilin and eosin (HE). Control slides that stain positively for the specific antigens were used to assure correct staining and stability of reagents used. Negative controls included the omission of the primary antibody.
Phase Contrast Microscopy
Growing cells on cover slips were observed with a phase-contrast microscope (Olympus IX50 with a PMC35Dx photo micrographic system).
Scanning Electron Microscopy
NG97 cells were grown to sub confluence on 13 mm round cover slip in complete medium. The cells were fixed with 2.5% glutaraldehyde and 4% paraformaldehyde in phosphate buffer (pH 7.4) for 1 hour at room temperature. Then, the cells were post-fixed in 1.0% osmium tetroxide (OsO4) for 10 minutes, washed in 0.1 M phosphate buffer (pH 7.2) and dehydrated in a grade series of ethanol. Cover slips were critically point dried using liquid CO2 as transition fluid. The specimens were cold sputter coated with gold and observed in a JEOL JMS 5800 LV scanning electron microscope (SEM) accelerating voltage of 10 kV.
Growth Curve
NG97 cells were collected from 13th passage for determination of growth curves. Briefly, semi confluent cultures were trypsinized and cells resuspended in complete medium for counting. Cells (1 × 104) were plated into each well of a 12-well plate and counts from triplicate wells were made daily for 10 days. Trypsinized cells were counted in hemacytometer chamber and numbers were averaged for each time interval. Cell population doubling time was calculated from the linear phase of the growth curve, and the saturation density was the plateau point on the growth curve after the linear growth phase.