Figure 1

Flow Cytometry Analysis of NCCM-Induced Protection from Apoptosis in NP Cells. (A) Representative sample of flow cytometry analysis of bovine NP cell death (Propridium Iodide (PI) (Y axis) and Annexin V (X axis) staining. All experiments were performed in triplicate. NCCM was generated from notochordal cells from six canine specimens. (a) 0% (basal medium) control, (b) IL-1β + FasL, (c) IL-1β + FasL + NCCM (b) The use of IL-1β + FasL causes an increase in the PI+ and Annexin V+ cells (upper right quadrant = dead cells) as compared to either (a) or (c). PI+ and Annexin V- = necrosis; PI + and Annexin V+ = apoptotic cell death. Treatment with NCCM (c) results in far fewer cells that are PI+ and Annexin V+ with an appearance that is almost equivalent to baseline (a). The black oval in (b) represents the cell population within the IL-1β + FasL treated cells that are 'rescued' by NCCM since these increasingly Annexin V+ and progressively PI+ cells are not apparent in the NCCM-treated groups. Triplicate experiments are presented in graphically in Figure 1 (b). (B) Summary of flow cytometry NP cell death assays (triplicate experiments as depicted in Figure 1 (a) above. The dark bars depict the percentage of all dead cells (Annexin V+ plus PI+) and the white bars depict the percentage of the total dead cells for each condition that died via apoptosis (Annexin V+ but PI-). There is little death in the untreated (0% (basal) media) cells but there is a marked increase in cell death when treated with IL-1β + FasL. This increase in both total cell death (from 2.5% to 8.8%) and apoptotic cell death (from 31.69% to 46.2%) is brought almost to baseline by the use of NCCM (apoptotic cell death is brought back to a level that is identical to the untreated cells). The differences between IL-1β + FasL and IL-1β + FasL + NCCM in terms of both total and apoptotic cell death are statistically significant at P = .016 and P = .0002 respectively (see asterisk indicating significance). Treatment with IL-1β + FasL + NCCM results in the most cell death as a function of apoptotic, not necrotic cell death. Abbreviations: IL-1β (Interleukin-1beta) FasL (Fas Ligand), NCCM (notochordal cell conditioned medium),