Therefore, the apoptotic nature of cell death was further verified by examining the biochemical differences observed in the Ad-eGFP or Ad-eGFP-PHB infected cells in presence or absence of STS by quantification of caspase-3-enzyme activity.
The activation of the different pathways of apoptosis generated by RBAc PDT was determined by quantification of active caspase-9 (37 and 35 kDa), caspase-8 (18 kDa) and caspase-3 (17 kDa), and cleaved caspase-12 (40 kDa) during 72 h of recovery after PDT.
Alternatively, apoptosis was determined by quantification of specific caspase-3 enzyme activity (fluorescence units/ μg protein) in protein lysates from cultured cells as described recently.
In addition to morphological assessment, activation of apoptosis was confirmed biochemically by quantification of cleaved-caspase 3. Briefly, hepatocytes in culture for 72 and 96 hours, treated or not with TGFβ, were fixed in acetone/methanol.
Furthermore, VSMC apoptosis, measured by quantification of cleaved caspase-3, was 43% lower in MMP-7 knockout mouse VSMCs compared with wild-type VSMCs following treatment with Fas-L.
Bar plots: quantification of caspase 3 signals corrected by the total cell number (by DAPI) and normalized to the HD controls (second bar).
Subsequently, cells were lysed and loaded with a luminogenic caspase-3/7 substrate and incubated for more 30 minutes at room temperature, followed by quantification of light emission as a function of caspase 3/7 activity.
Quantification of caspase 3, ALDH and F4/80 staining of tumor sections from 0%and10%0% MSC groups.
In soybean control cells a low level of caspase 3-like activity, measured by quantification of free p-nitroaniline (0.018 ± 0.002 mM pNA), and probably due to normal cell turnover, was detected (Fig. 5a).
Caspase 3-like activity was measured, after 30 min cell treatment, using the "caspase-3 colorimetric activity assay kit" (Chemicon International, Inc., Temecula, CA), as previously described [ 26] by quantification of free p-nitroaniline (pNA) released by the enzymatic cleavage of the caspase 3 synthetic substrate Ac-DEVD-pNA.
