PMC:17827 / 33119-35249 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/17827","sourcedb":"PMC","sourceid":"17827","source_url":"http://www.ncbi.nlm.nih.gov/pmc/17827","text":"Flow cytometry/histochemical analysis of cells negatively isolated from primary culture\nNegative isolation from primary culture using Dynabeads® M-450 CD14 (clone RMO52) resulted in cells that were Thy-1+ (on average, approximately 74%; n = 9; Fig. 2A and Table 1b) and, more importantly, prolyl-4-hydroxylase+ (on average, approximately 85%; n = 9; Table 1b) (mAb 3-2B12, Dianova), as shown by FACS analysis (Table 1b) and confirmed by immunohistochemistry in chamber slides. There were very few contaminating non-specific esterase+ (n = 3 RA and n = 3 OA patients; Fig. 2C), CD14+, CD68+, and/or CD11b+ macrophages (\u003c2%; Fig. 2B,D and Table 1b), as well as \u003c1% T cells (CD3+), B cells (CD19+/20+), plasma cells (CD38+), NK cells (CD56+), dendritic cells (CD83+), PMN (CD15), or endothelial cells (CD144+; von Wille-brand factor-positive).\nThe average yield of RA-SFB negatively isolated from primary culture was (2.8 ± 0.9) × 107 cells (mean ± SEM; n = 7; open synovectomy samples). A similar yield was observed upon isolation of cells from arthroscopic synovectomy samples ([1.7 ± 0.6] × 107 cells; n = 3). There was no significant difference between the yield from the primary culture of the RASM and that of the OA SM.\nInclusion of the trypsin component in the initial tissue digestion led to a considerably higher yield of cells following negative isolation from primary culture (1.2-fold to 5-fold; n = 3) than without trypsin or with DNAse instead of trypsin.\nThe composition of the cells was very similar to that obtained with primary-culture or first-passage normal skin-FB (Table 1g). Comparable results, although with a considerably lower percentage of Thy-1+ cells, were also obtained when OA-SFB were negatively isolated from primary culture (Table 1e). Conventional fourth-passage RA-SFB (Table 1d) or OA-SFB (Table 1f) showed FB markers on a high percentage of cells (\u003e98% prolyl-4-hydroxylase+ cells; \u003e80% Thy-1+ cells) and contained virtually no contaminating macrophages (\u003c2% CD14+-positive or CD68+-positive cells). The same was true for isolated primary RA-SFB kept in culture until fourth passage (Table 1c).","divisions":[{"label":"Title","span":{"begin":0,"end":87}}],"tracks":[]}