The caspase-9 activity assay by the caspase-Glo 9 kit (Promega, Shanghai, China) and mitochondrial membrane potential (MMP) reduction assay by the JC-1 dye (Invitrogen, Shanghai, China) were described in detail in previous studies [20 (link), 51 (link)].
Caspase glo 9 kit
Manufactured by Promega
Sourced in China, United States
The Caspase-Glo 9 kit is a luminescent assay that measures caspase-9 activity in cells. It provides a luminescent readout that is proportional to the amount of caspase-9 activity present in a cell lysate sample.
Automatically generated - may contain errors
3 protocols using caspase glo 9 kit
1
Caspase-9 Activity and Mitochondrial Potential
2
Measuring 3D Tumor Spheroid Viability
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Spheroid images in transmitted light were obtained using an inverted Axio Observer A1 microscope (Carl Zeiss, Gӧttingen, Germany) equipped with ×20/0.4 objective lens. Spheroid diameters were measured using the instrument software.
Cell viability in spheroids was measured by caspase activity assay using Caspase-Glo 9 kit (Promega, Madison, WI) according to manufacturer's manual with minor changes. Each sample contained 10–12 spheroids in PBS collected in one well of a 96-well plate. As a negative control (maximal cell viability), 2D cultured PANC-1 cells with confluency of 70% were used. The positive control (maximal caspase activity) was a sample with 3D PANC-1 spheroids after 48 h incubation with 20 μM of doxorubicin. Obtained values of luminescence, measured using 96-well plate spectrophotometer SpectraMax M2 (Molecular Devices, Sunnyvale, CA), were normalized by protein content, determined by Bradford assay.
Cell viability in spheroids was measured by caspase activity assay using Caspase-Glo 9 kit (Promega, Madison, WI) according to manufacturer's manual with minor changes. Each sample contained 10–12 spheroids in PBS collected in one well of a 96-well plate. As a negative control (maximal cell viability), 2D cultured PANC-1 cells with confluency of 70% were used. The positive control (maximal caspase activity) was a sample with 3D PANC-1 spheroids after 48 h incubation with 20 μM of doxorubicin. Obtained values of luminescence, measured using 96-well plate spectrophotometer SpectraMax M2 (Molecular Devices, Sunnyvale, CA), were normalized by protein content, determined by Bradford assay.
3
Caspase-9 Activation Assay in A549 Cells
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To perform the caspase-9 activation assay in A549 cells, a total of 104 cells were seeded in a 100 μl volume of DMEM media using a black clear bottom 96 well plate. After 24 h, cells were incubated with a final concentration of 50 μM of the conjugate prepared in PBS for 24 h. Subsequently caspase-9 activity was measured by the luminescence measurement on a Tecan M200 plate reader after incubation at RT for 1 h and adding 100 μl of the freshly reconstituted Caspase-9 reagent from the Caspase-Glo® 9 kit (Promega, Madison, WI, USA).
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