Etermination of the absence of DDD was based on macroscopic examination. Tissue samples were macroscopically dissected by the staff surgeon. Only IVD tissue from the convex side of the scoliotic disc was used for processing. To prevent crosscontamination between IVD-derived tissues, remnant endplate material was completely resected and a broad section of the transition zone extending well into RO5186582 web definedThe simian virus 40 large T antigen (SV40LTag) and human telomerase reverse transcriptase (hTERT) cDNAs were cloned into pBABE-hygro [16] and pBMN-IRESNEO vectors, respectively. Production of viral particles was performed as described previously [17]. Viral titres were sufficiently high to achieve nearly 100 infection. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26266977 Retrovirally transduced P5 AF and NP cells from donors 4 and 5 were selected with 400 g/ml G418 and 25 g/ml hygromycin B (PAA Laboratories/GE Healthcare Life Sciences, Somerset, UK). Immediately following selection, clones were generated by plating less than 1 cell (that is, ?.5) per well in 96-well plates in reduced selection medium (200 g/ml G418 and 12 g/ml hygromycin B). After 1 week of culture, small colonies became discernible. Culture wells with more than one colony were omitted from further study to ensure single picks. Single clones were expanded under continued antibiotic selection pressure and expanded for three more passages (P1 to P4) for cryogenic storage and screening experiments. Selected clones were expanded and remained stable for 8 months or 328 additional population doublings.van den Akker et al. Arthritis Research Therapy 2014, 16:R135 http://arthritis-research.com/content/16/3/RPage 3 ofTable 1 Intervertebral disc donor characteristics and clonesaMorphology Donor D1 D2 D3 D4 DaSex M F M F MAgeb, yr 8 13 14 15Medical indication Spina bifida/scoliosis Idiopathic scoliosis Spina bifida/scoliosis Idiopathic scoliosis Spina bifida/scoliosisPosition IVD T11-L4 L1-L4 T12-L4 T7-T10 T6-LT mRNA primary isolates Positive Negative Negative Negative NegativebNP clones N/A N/A N/A 34WaveCobblTiny19141IVD, Intervertebral disc; L, Lumbar; N/A, Not applicable; NP, Nucleus pulposus; T, Thoracic. Age in years at the time discs were obtained during surgery. Tissues were obtained from young adolescent scoliosis patients who had undergone correction surgery. In contrast to herniated or adult discs, these intervertebral discs showed no signs of degeneration; they had clear, lucid nuclei pulposi that could easily be distinguished from the annulus fibrosis. Each cell isolate (from donors D1 to D5) derived from AF and NP tissue excised at multiple adjacent levels, ranging from T6 to L4 as indicated, in a single individual. The total number of generated clones is indicated in column `Immortal NP clones’, and the amount of cobblestone (Cobbl), wavelike (Wave) and tiny clones is indicated in column `Clonal morphology’. Brachyury T (T) expression was measured in all primary isolates at the mRNA and protein levels; only D1 was found to be positive.Telomeric repeat amplification protocolRNA isolation and quantitative real-time PCRTo measure TERT activity, a telomeric repeat amplification protocol assay was performed according to the manufacturer’s instructions (TRAPeze Telomerase Detection Kit; Millipore, Amsterdam, the Netherlands) [18]. Briefly, 1 g of total protein input was used to produce tandem TAAGGG repeats (extension step at 30 for 30 minutes); these repeats were amplified by PCR using telomere-specific primers (30 cycles).