Figure 3

Chondrocyte death consists of apoptosis and necrosis. (a) Morphologic analysis of chondrocyte apoptosis and necrosis. The nuclei of apoptotic cells were labeled by TUNEL with green fluorescence whereas the nuclei of necrotic cells were labeled by PI with red fluorescence (donor age 63 years). All cell nuclei were labeled with blue Hoechst dye. Results are representative of three OA cartilage samples. (b) The rates of cell apoptosis and necrosis quantified by flow cytometry. Apoptotic cells were labeled by annexin V whereas necrotic cells were labeled by PI at the same time. Total events are 10,000. The x axis represents FL1-H (log) with green fluorchrome for annexin V labeling, and the Y axis represents FL2-H (log) with red fluorchrome for PI labeling. The percentage of cells that are single or double positive for annexin V and PI is indicated in each grid. The upper left grid represents the number of PI single positive cells. The lower right grid represents the number of annexin V single positive cells. The upper right grid represents the number of cells positive for both PI and annexin V. The number of cells that are single positive for PI is 9.6% for anti-CD95, and 3.9% for control, SB, and Anti-CD95+SB. Anti-CD95: treatment with a mAb anti-Fas CH 11 (100 ng/ml) in serum-free medium for 17 hours. SB: treatment with SB203580 (10 μmol/l) for 17 hours. SB+Anti-CD95: treatment with SB203580 for 2 hours followed by anti-Fas treatment for 17 hours. Control: cells were treated with DMSO only. Results are representative of five OA cartilage samples. (c) FACS analysis of OA chondrocytes labeled by annexin V and PI distinguishes apoptosis from necrosis. Chondrocytes were labeled into four groups by Annexin-V-FLUOS Staining Kit: x axis is FL1-H (log), and fluorchrome is green for annexin V (panel b [lower right] and panel c [lower right]); and y axis is FL2-H (log), and fluorchrome is red for PI (panel b [upper left] and panel c [upper left]). Living cells are shown to the lower left and double staining to the upper right of panels b and c. DMSO, dimethyl sulfoxide; FACS, fluorescence-activated cell sorter; mAb, monoclonal antibody; OA, osteoarthritis; PI, propidium iodide; TUNEL, terminal dUTP nick-end labeling.