FLG) described previously [31,32,36]. U87 cells expressing firefly luciferase-eGFP (U87-Luc) were sorted to purity based on GFP expression, expanded and stored at 2150 degrees Celsius. All experiments were performed with U87-Luc cells at less than passage 15 (approx 4 months), post their acquisition from ATCC. U87 cells were maintained in DMEM supplemented with 10% fetal bovine serum. GBM – endothelial cell co-cultures Co-localization studies. Fifteen thousand HBMEC or HUVEC cells were plated on coverslips (35 cm2) coated with Matrigel extracellular matrix (BD Biosciences, San Jose, CA) as per manufacturer’s instructions and then grown in EGM-2MV. After 24 hours, either 15,000 U87 cells or 30,000 primary GBM cells per 35 mm2 were added to the cultures and grown in Serum Free DMEM. Unless mentioned otherwise, co-localization measurements were performed 24 hours following addition of tumor cells. Growth assays. Growth assays were conducted with Matrigel-coated, 96 well tissue culture plates containing 3000 HBMECs and 3000 U87 or 6000 primary GBM cells per well, plated and grown as described above. Primary Human GBM Tissue Formalin-fixed, paraffin-embedded archival specimens of GBM were retrieved from the pathology files at Washington University School of Medicine in accordance with an Institutional Review Board–approved protocol for human research. Overexpression and knockdown For knockdown experiments, CXCL12 or CXCR4 specific shRNAs (CXCL12: TGTGCATTGACCCGAAG CTAA or G- AGTACCTGGAGAAAGCTTTA; CXCR4: GCTGCCTTAC- TACATTGGGAT) or a scrambled (control) shRNA sequence (CCGGCAACAAGATGAAGAGCACCAA) cloned into a lenti- viral packaging vector (pLKO.1) were obtained from the Genome Institute at the Washington University School of Medicine in St Louis. Viral particles were produced from each packaging vector separately by the Viral Vectors Core Facility of The Hope Center for Neurological Diseases at Washington University School of Medicine. HBMECs were infected with lentivirus encoding either control or CXCL12 shRNA and U87 cells were infected with lentivirus encoding either control or CXCR4 shRNA as previously described [37]. Cells expressing shRNA constructs were selected using puromycin. As described in the results, knockdown of CXCL12 expression levels were determined by PCR and ELISA analysis. Knockdown of CXCR4 was confirmed by western blot. For over-expression experiments, HUVECs were infected with lentivirus containing CXCL12 gene as described previously [38]. CXCL12 overexpression was confirmed by ELISA. Quantitative PCR RNA was isolated from endothelial cells using the RNeasy system (Qiagen (Valenica, CA)). Copy DNA was synthesized from 100 ng of RNA using iScript RTase, and CXCL12 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts were amplified using the power SYBR GREEN PCR Master Mix (Applied Biosystems (Carlsbad, CA)) according to the manufac- turer’s instructions. Primers for CXCL12 (forward primer, A- TGCCCATGCCGATTCTT; reverse primer, GCCGGGCTA- CAATCTGAAGG) and GAPDH (forward primer, GGCAAA- TTCAACGGCACAGT; reverse primer, AGATGGTGATGGG- CTTCCC) were obtained from Integrated DNA Technologies CXCL12 Mediated GBM-Endothelial Cell Interactions (Iowa City, IA) and used at 300 nmol/L. Samples were run in triplicate with a corresponding GAPDH control for each sample. PCR and data collection were done using the BioRad Mini- Opticon Real Time PCR machine and Opticon Monitor 3 Software from BioRad (Hercules, CA). Relative transcript copy number for each CXCL12 and corresponding GAPDH sample were calculated using the delta-delta-C(t) method. The CXCL12 relative expression values for each condition were normalized to that of the lowest relative expression level for each experiment (n = 3). CXCL12 ELISA HBMEC or HUVECs (wild type or infected with viruses as described in results) were plated onto gelatin or Matrigel coated dishes and maintained in EGM-2MV media for 24 hours. The media was then changed to Serum Free DMEM. After 48 hours of additional incubation, culture supernatants were collected and concentrated (1006) for CXCL12 detection by indirect ELISA assay. Briefly, CXCL12 standards, culture supernatants from endothelial cells and DMEM from Matrigel alone were incubated in a 96-well plate overnight in 20 mM sodium bicarbonate buffer (pH 9.5). The plate was blocked with 2% BSA for 1 hour and then incubated with rabbit anti-hCXCL12 (1:200, PeproTech Inc.) for 1 hour. OPD substrate (Sigma-Aldrich) was added to the plate following incubation of goat anti-rabbit IgG HRP (1:2500, Bio- Rad) for 1 hour. The reaction was stopped by 3M HCL and OD at 490 nm was measured with a microplate reader (Bio-Tek Instruments, Inc.). CXCL12 concentrations were calculated with reference to an ELISA standard curve. All samples were analyzed in duplicate. Western blot analysis Protein extracts were obtained by lysing cells with lysis buffer [20 mmol/L Tris (pH 7.4), 137 mmol/L NaCl, 10% glycerol, and 1% Triton X-100] supplemented with Complete Protease Inhibitors (Roche) and Phosphatase Inhibitor Cocktail set #IV (Calbiochem (Gibbstown, NJ)). The proteins (25 mg) were resolved with 10% Bis-Tris gels (Invitrogen) and transferred onto the Hybond ECL nitrocellulose membrane (Amersham (Piscataway, NJ)) according to standard protocols. Blots were then probed with polyclonal anti-CXCR4 antibody. Total protein loading per lane was evaluated with anti-b-Actin antibody. This was followed by incubation with IRDyeH conjugated secondary antibodies (LI- COR (Lincoln, NE)). Blots were imaged with the Odyssey fluorescent scanning system (Li-Cor). Immunofluorescence Cells were fixed with 70% ethanol for 10 minutes at 220uC. Immunostaining of CXCL12, GFAP or CD31/PECAM was done as described [14]. CXCR7 antibody 11G8 was used (15 mg). Secondary AlexaFluor 488 or 568–conjugated donkey anti-mouse, AlexaFluor 555-conjugated donkey anti-rabbit, AlexaFluor 488– conjugated donkey anti-rat or anti-goat antibody were used at a concentration of 1:1750 (Molecular Probes) for 90 minutes. Nuclei were counterstained with DAPI (4N, 6-diamidino-2-phenylindole) or TO-PRO 3 (Invitrogen). Tissue sections and immunohistochemistry Sections (5 mm) were deparaffinized in xylene and rehydrated in descending alcohols to water. Endogenous peroxidase was blocked with 3% H2O2 in TBST [10 mmol/L Tris (pH 8.0), 0.15 mol/L NaCl, 0.05 Tween] and nonspecific avidin/biotin binding sites were blocked with the Vector Avidin/Biotin Blocking kit (Vector PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e33005