Amoureux et al. BMC Cancer 2010, 10:91 http://www.biomedcentral.com/1471-2407/10/91 of NCAM [17]. Immunostaining was analyzed by using a semi-quantitative score. Briefly, the percentage of posi- tive tumor cells per slide (0% to 100%) was multiplied by the main intensity of staining (0: negative; 1: very weak; 2: weak; 3: moderate; 4: intense). PSA-NCAM expression score ranged from 0 to 320 in GBM. It was up to 360 in MB. Preparation of tissue and cell extracts, and protein measurement for ELISA Cells or tissues were homogenized in PBS, 0.1% Tri- tonX-100 and sonicated. The nuclear and mitochondrial fractions and debris were removed by centrifugation at 10000 g, 4°C, for 15 minutes. Total proteins were mea- sured from the supernatant using the BCA kit (Perbio, Brebieres, France). Samples were initially diluted in phosphate buffered saline (PBS) at 37 μg/ml of protein and tested in the ELISA assay. If necessary, the dilution was adjusted to be in the linear range of the standard curve, and the PSA-NCAM concentration calculated in pg PSA-NCAM/μg of total protein. Purification of PSA-NCAM for ELISA standard curve, and of NCAM for anti-NCAM antibody generation PSA-NCAM was purified from postnatal stage P0 mice by affinity chromatography using the previously charac- terized H28 monoclonal antibody [18] immobilized on an Aminolink column (Perbio) at 10 mg/ml of resin, according to the manufacturer’s procedure. Protein con- centration was measured using the BCA kit. NCAM was purified from adult mice brain on the same affinity column, and used to produce a goat poly- clonal anti-NCAM antibody (Eurogentec, Orleans, France). The anti-NCAM recognizes the 3 main NCAM isoforms (data not shown). PSA-NCAM ELISA Ninety six-well microtiter plates (Nunc Maxisorp, VWR, Strasbourg, France) were coated with 100 μl of mouse monoclonal antibody directed against PSA- NCAM (1/4000 in PBS) (Abcys). After PSA-NCAM capture, the wells were washed 3 times with PBS (Sigma, Lyon, France) and blocked with 200 μl of PBS containing 5% bovine serum albumin (BSA; Sigma) for 2 h at 37°C. Hundred μl of samples of unknown PSA- NCAM concentration, and standards were then incu- bated overnight at 4°C. Standards were prepared from known amounts of PSA-NCAM purified from postna- tal P0 mice as described above. The dose range for PSA-NCAM standard curve was 0.25 to 16 ng/ml. After 5 washes with PBS containing 0.1% Tween20 (PBST), wells were incubated with 100 μl of goat anti N-CAM prepared from purified mouse NCAM as described above (1/4000; Eurogentec) in PBST, 5% Page 3 of 12 BSA (PBSTB) for 2 h at 37°C. After 5 washes with PBST, wells were incubated with 100 μl of donkey horseradish peroxidase (HRP)-coupled anti-goat anti- body (Jackson Laboratory, Ban Harbor, MA) diluted 4000 times in PBSTB for 2 h at 37°C. Plates were washed 5 times with PBST and incubated with 100 μl of Tetramethylbenzidine (TMB; Sigma). TMB tablets were diluted in 0.2 M dibasic sodium phosphate, 0.1 M citric acid, 0.6% H2O2. The reaction was stopped with 100 μl 1.8 N H2SO4. Absorbance was read at 450 nm using a 96-well plate reader (Berthold, Thoiry, France) and the concentration of PSA-NCAM in sam- ples was deduced from the standard curve. For ELISA validation, the rhabdomyosarcoma TE671 cell line was used and grown as previously described [17]. The specificity of the ELISA test was established using endoN. A TE671 extract was digested with endoN for 4 h at 37°C. Non digested and digested extracts were serially diluted and tested in parallel in the same ELISA test to ensure that the signal detected with the ELISA was indeed specific for PSA-NCAM. Cell culture, endoneuraminidase treatment and immunocytochemistry The rat C6 glioma cell line (ATCC-LGC, Molsheim, France) was grown in Dulbecco’s modified Eagle’s med- ium (DMEM) supplemented with 10% fetal calf serum (FCS), penicillin (50 Units/ml) and streptomycin (100 μg/ml). The C6-PSA-NCAM cell line (generous gift of M. Fukuda, The Burnham Institute for Medical Research, La Jolla, CA) over expresses PSA-NCAM after transfection with a polysialyltransferase construct (ST8- SiaIV/PST) and has been previously described [19]. C6- PSA-NCAM cells were grown in DMEM supplemented with 10% FCS, penicillin, streptomycin and geneticin (0.5 mg/ml). Both cell lines display similar proliferation rates [20]. For olig2 quantification, 10 mm diameter plates of C6 and C6-PSA-NCAM were used when cells had reached confluence. The GBM9 cell line was described in our previous study [9]. Cells were grown as neurospheres in Neuro- basal medium supplemented with B27, GlutaMAX™, EGF (20 ng/ml) and bFGF (20 ng/ml), both from Pepro- tech (London, UK). For splitting cells, spheres were spun down (300 g, 5 minutes, RT) and dissociated with 1 to 2 ml Accumax (Abcys, Paris, France) for 30 min- utes at 37°C. Single cells suspensions were obtained by mechanical dissociation. Cells were subsequently spun down and resuspended in growth medium. For differen- tiation experiments, cells were plated in Poly-D-lysine (10 μg/ml) coated multiwell-24 plates (30000 cells/well). All cells were grown in humidified atmosphere contain- ing 5% CO2 at 37°C. All tissue culture reagents were from Invitrogen (Cergy Pontoise, Fance).