Table 3.
Binding of annexin-V and propidium iodide to WT vs. Adam8−/− eosinophils do not differ when the extrinsic apoptosis pathway is triggered in vitro
| Time point | WT EosinophilsA | Adam8−/− Eosinophils | WT Eosinophils | Adam8−/− Eosinophils | WT Eosinophils | Adam8−/− Eosinophils |
|---|---|---|---|---|---|---|
|
| ||||||
| % Apoptotic Cells Mean (SEM) n |
% Apoptotic Cells Mean (SEM) n |
% Late Apoptotic Cells Mean (SEM) n |
% Late Apoptotic Cells Mean (SEM) n |
% Viable Cells Mean (SEM) n |
% Viable Cells Mean (SEM) n |
|
|
| ||||||
| 0 h | 0 (2.9) 3B | 0 (3.1) 3 | 0.5 (0.3) 3 | 0 (0.2) 3 | 0.3 (2.7) 3 | 0.2 (3.1) 3 |
| 2 h | 4.1 (6.4) 3 | 8.4 (10.6) 3 | 0.3 (0.1) 3 | 1.2 (0.5) 3 | 3.6 (6.3) 3 | 8.3 (10.7) 3 |
| 4 h | 19 (5.9) 3 | 16.7 (6.1) 3 | 3 (1.3) 3 | 7.3 (0.7) 3 | 18.3 (5) 3 | 19.4 (7) 3 |
| 6 h | 12.2 (9.3) 3 | 9.9 (19.3) 3 | 5.8 (2.5) 3 | 10.9 (8.4) 3 | 16.4 (8.2) 3 | 30.1 (7.8) 3 |
| 8 h | 19.7 (15) 3 | 7.3 (2.2) 3 | 7.5 (1.7) 3 | 8.2 (0.7) 3 | 30.2 (8.9) 3 | 29.5 (6.4) 3 |
| 20 h | 46.2 (10.9) 3 | 40.2 (9.6) 3 | 38.7 (12.5) 3 | 40.6 (8.9) 3 | 57.2 (2.1) 3 | 51.8 (2.6) 3 |
Eosinophils and macrophages were isolated from unchallenged BALB/c WT and BALB/c Adam8−/− mice, as described in Methods. Equal numbers of eosinophils or macrophages were incubated with a cross-linking antibody to CD95 (FAS) which activates this death domain-containing receptor, or a non-immune isotype control antibody for up to 20 h for eosinophils or 48 h for macrophages. At intervals, cells were stained with annexin-V conjugated to Alexa 488 and propidium iodide, and the percentage of cells staining positively and negatively with these reagents was quantified using flow cytometry. The results for cells incubated with a non-immune control antibody were subtracted from those incubated with anti-CD95. We determined the percentages of apoptotic cells (cell staining positively with annexin-V and negatively with propidium iodide), late apoptotic cells (cells staining positively with both annexin-V and propidium iodide), and viable cells (cells not stained with either annexin-V or propidium iodide).
Data are mean ± SEM; n = number of independent experiments.
We were not able to induce significant positive staining for either annexin V or propidium iodide in either WT or Adam8−/− macrophages by treating cells with the FAS cross-linking antibody for up to 48 h.