The HeLa-Vehicle Standard was prepared using HeLa cells treated for 4 hours with vehicle (DMSO). Background-subtracted data values (mean +/- SD) are graphed.
The HeLa-Staurosporine Standard was prepared using HeLa cells treated for 4 hours with 1 µM staurosporine (ab120056). Background-subtracted data values (mean +/- SD) are graphed.
Cells were treated with vehicle-DMSO (A and C) and 1 µM staurosporine (B and D) for 4 hours. Extracts prepared from cell pellets were analyzed in triplicates. Raw pro-caspase 3 signals (solid line) recorded at 360/450 (excitation/emission) nm and active-caspase 3 signals (dashed line) recorded at 555/595 nm are shown. Dotted lines represent no material background signal.
Lysates corresponding to 0.33x10ab120056) in a 96-well plate. 100 µL of the lysates were analyzed in triplicates. Background-subtracted pro-caspase 3 (solid line) and active-caspase 3 (dashed line) signals are shown in left panel. Relative pro-caspase 3 (solid line) and active-caspase 3 (dashed line) concentrations interpolated from appropriate standard curves and expressed as percent of maximal signals are shown in right panels. Staurosporine EC50 of pro-caspase 3 was 0.9 µM. Staurosporine EC50 of active-caspase 3 was 1.1 µM.
Cell extracts were prepared from 4 hours vehicle-treated (lanes 1 and 3) and 1 µM staurosporine-treated (lanes 2 and 4) Jurkat cells. Extracts were incubated with the Caspase 3 Microplate, captured proteins were extracted and analyzed by Western blotting using a pro+p17 caspase 3 antibody ab32351 (Lanes 3 and 4). 25% of the amounts of the extracts used for immunocapture were also analyzed directly by the Western blotting (lanes 1 and 2) with the same antibody. Note that the Caspase 3 Microplate captures both the pro- and p17 subunit of caspase 3.
Cells were treated for 4 hours with 1 µM staurosporine or drug’s vehicle (DMSO). HeLa cell extracts (400, 200 and 100 mg/mL, graphed left to right) or Jurkat cell extracts (50, 25 and 12.5 mg/mL, graphed left to right) were analyzed using this kit. Relative pro-caspase 3 concentrations were interpolated from HeLa-Vehicle standard curve and expressed as percent of vehicle-treated HeLa cell. Relative active caspase 3 concentrations were interpolated from HeLa-Staurosporine standard curve and expressed as percent of staurosporine-treated HeLa cell. Note that (1) staurosporine treatment reduced the pro-caspase 3 levels in both cell lines, (2) staurosporine treatment strongly induced the active caspase 3 levels in both cell lines, (3) Jurkat cells contained approximately 6 times higher caspase 3 concentrations (per total protein mass) compared to HeLa cells.