Acutase

Bone marrow cells were flushed out of the long bones of the mice and the cell suspension cultured in αMEM supplemented with 10% FCS, Pen/Strep and 100 ng/ml M-CSF (Prospec Bio) for three days. Next the non-adherent cells were removed by washing the cultures with PBS, and the adherent BMDMs harvested using Acutase (Sigma) or cell dissociation buffer (Enzyme-Free; PBS-based) (Gibco, Life Technologies). For osteoclast formation experiments, cells were plated in 96-well plates at 15 × 10³ cells in 100 µl medium supplemented with 25 ng/ml M-CSF and up to 100 ng/ml RANKL (a kind gift of Dr. J. Dunford, University of Oxford) per well. The culture medium was refreshed at day 2 and day 4 and the cells fixed on day 5 with 4% buffered formalin and stained for TRAP. TRAP positive cells containing three or more nuclei were counted as osteoclasts. For RNA isolation, BMDM cells were seeded in 6-well plates at 5 × 10⁵ cells per well in medium supplemented with 25 ng/ml M-CSF only (for macrophage cultures) or 25 ng/ml M-CSF plus 100 ng/ml RANKL (for osteoclast cultures) and cultured for 5 days as described above. For Western Blot analysis, BMDM cells were seeded in 6-well plates at 5 × 10⁵ cells per well in medium supplemented with 25 ng/ml M-CSF and cultured for 3 days.

Human serum, M-CSF, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), acutase, and ovalbumin was from Sigma. Heparin (5000 U/ml) was obtained from Leo Pharma, Denmark and complete protease inhibitor tablets was from Roche.
Proteins: Recombinant wild-type SOD3 and SOD3 containing a C-terminal c-Myc tag (SOD3-cMyc) was produced as previously described [42 (link)]. For the generation of the SOD3-cMyc expression plasmid, we used the forward primer (5′-TATACAGCTAGCATGCTGGCGCTACTGTGTTCC-3′) and a reverse primer encompassing the cMyc tag (underlined) (5′-TATGAATTCTCACAGATCCTCTTCTGAGATGAGTTTTTGTTCGGCGGCCTTGCACTCGCTCTC-3′) and cloned the product into the pIRES vector. The sequence of the obtained plasmid was verified by sequencing. mAb 5G8D4 and 7F6D9 anti-human SOD3 (GenScript) and rabbit anti SOD3 antisera (Davids Biotechnologie) were developed using recombinant human SOD3 as antigen and antibodies recovered by using protein G-Sepharose. Recombinant human and murine IFNγ was obtained from Invitrogen. ovalbumin was from Sigma. Human receptor-associated protein (RAP) was obtained from ENZO life sciences (BML-SE552). HRP-conjugated goat anti-rabbit Ig was from DAKO. Flow cytometric analyses were performed using PE-conjugated anti-CD14 (B&D Biosciences), APC-conjugated anti-CD4 (B&D Biosciences) or APC-conjugated mAb5G8D4 anti-SOD3.

Fresh human GBM material was acquired from 4 GBM surgical patients and cultured as previously reported
[9 (link),13 (link)]. Briefly, the dissociated tissue was washed, filtered through a 30 μm mesh and plated onto ultra-low adherence flasks at a concentration of 500,000 to 1,500,000 viable cells/ml. The stem cell isolation medium included human recombinant EGF (20 ng/ml), human bFGF (10 ng/ml) and heparin (2 μg/ml). Sphere cultures were then passaged by dissociation, washed, resuspended in neural stem cell culture medium (#05750,Stemcell Technologies), and plated on ultra low-adherence 96 well plates at 2000 cells per well for all subsequent drug testing.
Alternatively, patient-derived dissociated GBM tissues were plated onto laminin-1 coated plates (Sigma, 3-5 μg/ml). Cell populations were dissociated using Acutase (Sigma) and expanded for 5-10 passages, then plated on flat bottom for drug testing.