Caspase-3/9 activity was assayed using the Caspase-3/9 activity assay kit (Beyotime, Beijing, China) according to the manufacturer's instructions. After transfection, the cells were washed with cold PBS and centrifuged at 800× g. The cell pellet was then lysed by the addition of 50 μL of chilled cell lysis buffer and incubation on ice for 10 mins. The lysed mixture was clarified by centrifugation at 10,000× g for 1 mins and the supernatant was transferred to a new microcentrifuge tube. The protein concentration in the supernatant of each sample was measured using the Bradford assay and then adjusted to 100 μg of protein per 50 μL of cell lysis buffer for subsequent application to each well of a 96-well plate. Next, 50 μL of 2× reaction buffer containing dithiothreitol at a final concentration of 10 mM was added into each sample well. After mixing, the respective substrate of each caspase was added to each well and the plate was incubated at 37°C for 1–2 hrs. Finally, the absorbance of each reaction at 400–405 nm was measured on a microplate reader. Each experiment was performed in triplicate.
Caspase 3 9 activity assay kit
Manufactured by Beyotime
Sourced in China
The Caspase 3/9 Activity Assay Kit is a laboratory tool designed to measure the activity of the caspase-3 and caspase-9 enzymes, which are critical regulators of apoptosis, or programmed cell death. The kit provides the necessary reagents and protocols to quantify the enzymatic activity of these caspases in cell or tissue samples.
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9 protocols using caspase 3 9 activity assay kit
1
Quantifying Caspase-3/9 Activity in Cells
2
Caspase-Mediated Apoptosis Inhibition by Copper Complex
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The activity of caspase3, caspase9 was detected by (Caspase 3/9 Activity Assay Kit, Beyotime Biotechnology, Shanghai, China) following its instructions. Shortly, the HeLa cells were cultures in 96-well plate and incubated with [Cu(PMPP-SAL)(EtOH)] for 24 h. Then, the cells were treated with suitable caspase9 and 3 reagents and then incubated at 37 °C for 2 h. The optical density for caspase9 and 3 were measured using reader at 405 nm.
To further determine whether the inhibitory effect [Cu(PMPP-SAL)(EtOH)] on HeLa cells growth was affected by caspase pathway, HeLa cells were seeded at a concentration of 1 × 105 cells/mL in a 96-well plate, and 5 μM caspase pan-inhibitor (Z-VAD-FMK) was added to each well. After incubation for 0.5 h, HeLa cells were treated with [Cu(PMPP-SAL)(EtOH)] and continued to be cultivated for 24 h. Then cells were incubated with 20 μL of MTT for 4 h, and the OD at 570/655 nm were detected by microplate reader.
To further determine whether the inhibitory effect [Cu(PMPP-SAL)(EtOH)] on HeLa cells growth was affected by caspase pathway, HeLa cells were seeded at a concentration of 1 × 105 cells/mL in a 96-well plate, and 5 μM caspase pan-inhibitor (Z-VAD-FMK) was added to each well. After incubation for 0.5 h, HeLa cells were treated with [Cu(PMPP-SAL)(EtOH)] and continued to be cultivated for 24 h. Then cells were incubated with 20 μL of MTT for 4 h, and the OD at 570/655 nm were detected by microplate reader.
3
Caspase-3/9 Activity Assay Protocol
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After culture, NP cells were washed with PBS for two times and lysed using 300 μl lysis buffer provided by the test kit (Caspase-3/9 Activity Assay Kit, Beyotime, China) for 10 min. After centrifugation (15,000 rpm, 12 min, 4°C), the supernatant sample was collected and used to perform the chemical reaction according to the method described in the manufacture’s instructions. Finally, their activities were calculated according to the standard curve and the absorbance at a wavelength of 405 nm.
4
Apoptosis and Caspase Activation Assay
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For the determination of the apoptotic rate, an Annexin V-FITC Apoptosis Detection Kit (Vazyme, Nanjing, China) was used for the flow cytometry. After 48 hpi, PK-15 cells were trypsinized and collected in HBSS, where 400 μL 1× binding buffer was added to re-suspended cells for each sample. The cells were stained with 5 μL Annexin V-FITC and 5 μL propidium iodide (PI) for 10 min in the dark at room temperature. At least 1 × 104 cells in each sample were measured to identify the apoptotic cell populations.
To detect whether virus infection activated caspase 3 and caspase 9, the Caspase 3/9 Activity Assay Kit (Beyotime) was used. The kit contains Ac-DEHD-pNA and Ac-LEHD-pNA as caspase 3 and 9 specific substrates, respectively. The active caspases could cleave substrates to become fluorescent (excitation at 485 nm and emission at 530 nm).
To detect whether virus infection activated caspase 3 and caspase 9, the Caspase 3/9 Activity Assay Kit (Beyotime) was used. The kit contains Ac-DEHD-pNA and Ac-LEHD-pNA as caspase 3 and 9 specific substrates, respectively. The active caspases could cleave substrates to become fluorescent (excitation at 485 nm and emission at 530 nm).
5
Caspase-3/9 Activity Assay for Apoptosis
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Cell apoptosis was also assayed by a caspase-3/9 activity assay kit (Beyotime Institute of Biotechnology; cat no. C1115). In brief, whole cell protein was lysed and extracted as aforementioned. Then, 100 µg protein from each sample was incubated with 50 µl reaction buffer and 5 µl Ac-DEVD-pNA (4 mM) substrate at 37°C for 2 h. The absorbance at 405 nm was determined using a microplate reader (Thermo Fisher Scientific, Inc.).
6
Caspase-3 and Caspase-9 Activity Assay
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Caspase-3 and caspase-9 activities were measured using 30 μg protein obtained from extracted cells and Caspase-3/9 Activity Assay Kit (Beyotime Institute of Biotechnology; China; catalog no. C1115 and C1158) following the manufacturer’s instructions. Cells were incubated with Ac-DEVD-pNA (caspase-3 substrate) and Ac-LEHD-pNA (caspase-9 substrate) at 37°C for 2 h. Optical densities were determined at 405 nm using a colorimetric microplate reader. The relative caspase activity was recorded as the ratio to that of the control group.
7
Caspase-3/-9 Activity Assay
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Next, caspase-3/-9 activities were determined using a Caspase-3/-9 Activity Assay Kit (Beyotime, Beijing, China) according to the manufacturer's protocol. Absorbance at a wavelength of 405 nm was measured in a microplate spectrophotometer (Thermo Labsystems, Vantaa, Finland).
8
Caspase-3/9 Activity Assay in Oxaliplatin-Treated Cells
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After treatment with oxaliplatin in the experimental and control groups, respectively, cells were collected and caspase-3/9 activity was detected using caspase-3/9 activity assay kits (Beyotime) according to the manufacturer’s protocol.
9
Measuring Caspase Activity in Cells
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Caspase 3/9 activity assay kits (Beyotime Institute, Jiangsu, China) were used to detect caspase activities according to the manufacturer’s protocol. Briefly, cells were plated in 6-well plates for 12 h, treated with agents for 24 h, harvested and resuspended in lysis buffer. Cell protein was normalized, and caspase activities were evaluated by interaction between substrates and enzymes.
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