The activities of Caspase-3 and Caspase-9 are important markers of apoptosis process in cells. After treatment with 2.5, 5, 10 and 20 μM EMB for 6 h, the QSG7701 cells were collected and washed two times. The activities of caspase-3/-9 were determined using the Caspase-3/-9 activity kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Then, the reading of absorbance was taken at 405 nm by a Synergy H1 microplate reader.
Caspase 3 9 activity kit
Manufactured by Beyotime
Sourced in China
The Caspase-3/9 activity kit is a laboratory tool designed to measure the activity of the caspase-3 and caspase-9 enzymes. Caspase-3 and caspase-9 are critical mediators of programmed cell death, or apoptosis. The kit provides a quantitative, colorimetric method to assess the activity of these caspases in cellular or tissue samples.
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Lab products found in correlation
Ripa buffer, by Beyotime (2 mentions)
Bca assay, by Beyotime (2 mentions)
Synergy h1, by Synergy Software (1 mentions)
Annexin 5 fitc pi double staining apoptosis detection kit, by BD (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Facscalibur, by BD (1 mentions)
Bca method, by Beyotime (1 mentions)
Ldh assay, by Beyotime (1 mentions)
Ldh release detection kit, by Beyotime (1 mentions)
Model 680 microplate reader, by Bio-Rad (1 mentions)
14 protocols using caspase 3 9 activity kit
1
Caspase-3 and Caspase-9 Activity Assay
2
Caspase-3/9 Activity Quantification
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Caspase-3/9 activity in cell lysates was determined using a Caspase-3/9 activity kit (Beyotime Institute of Biotechnology, Nantong, China) according to the manufacturer's protocol. The caspase-3/9 activity was normalized by the protein concentration of the corresponding cell lysate and expressed as percentage of treated cells to that of control.
3
Apoptosis Assessment and Caspase Activity
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Seventy-two hours after transfection, apoptotic cells were stained with an Annexin V-FITC/PI double staining apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA). Apoptotic cells were assessed by flow cytometry (BD FACSCalibur, BD Biosciences).
At 72 h after transfection, cells were lysed in RIPA buffer (Beyotime Biotechnology, Co., Ltd.) and protein content was measured using BCA assay (Beyotime Biotechnology, Co., Ltd.). Equal amounts of protein were used to determine caspase-3/9 activity using a caspase-3/9 activity kit (Beyotime Biotechnology, Co., Ltd.). Optical density (OD) value was read using a microplate reader (Bio-Rad Laboratories) at 405 nm.
At 72 h after transfection, cells were lysed in RIPA buffer (Beyotime Biotechnology, Co., Ltd.) and protein content was measured using BCA assay (Beyotime Biotechnology, Co., Ltd.). Equal amounts of protein were used to determine caspase-3/9 activity using a caspase-3/9 activity kit (Beyotime Biotechnology, Co., Ltd.). Optical density (OD) value was read using a microplate reader (Bio-Rad Laboratories) at 405 nm.
4
Colorimetric Assay of Caspase-3 and -9 Activity
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The activity of caspase-3 and caspase-9 was determined using the caspase-3/-9 activity kit (Beyotime Institute of Biotechnology, Haimen, China) based on a colorimetric assay of the yellow formazan chromophore p-nitroaniline (pNA) after cleavage from the labeled substrate DEVD-pNA or LEHD-pNA. To evaluate the activity of caspase-3 or caspase-9, cells were homogenized in 80/100μl reaction buffer (1% NP-40, 20 mM Tris-HCl (pH 7.5), 137 mM NaCl, and 10% glycerol) containing 10 μl caspase-3 substrate (Ac-DEVD-pNA, 2 mM) or caspase-9 substrate (Ac-LEHD-pNA, 2 mM) after all treatments. Lysates were incubated at 37°C for 2 h. Samples were measured with an ELISA reader (Multiskan Ascent) at an absorbance of 405 nm. The detailed analysis procedure is described in the ELISA manufacturer's protocol. The caspase activity, normalized for total proteins of cell lysates, was then expressed as fold of the baseline caspase activity of control INS-1 cells.
5
Caspase-3/9 Activity Assay
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Caspase-3/9 activity in cell lysates was determined using a caspase-3/9 activity kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. Caspase-3/9 activity was normalized by the protein concentration of the corresponding cell lysate and expressed as percentage of treated cells to that of control.
6
Caspase 3/9 Activity Quantification
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Caspase 3/9 activity in cell lysates was determined using a Caspase 3/9 activity Kit (Beyotime Biotech, China) according to the manufacturer's protocol. The caspase 3/9 activity was normalized by the protein concentration of the corresponding cell lysate and expressed as percentage of treated cells to that of control.
7
Caspase-3/9 Activity Quantification
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Caspase-3/9 activity in cell lysates was determined using a Caspase-3/9 activity kit (Beyotime Institute of Biotechnology, Nantong, China) according to the manufacturer's protocol. The caspase-3/9 activity was normalized by the protein concentration of the corresponding cell lysate and expressed as percentage of treated cells to that of control.
8
Caspase 3/9 Activity Assay Protocol
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Caspase 3/9 activity was measured spectrophotometrically as described previously (Liu et al., 2017a). Caspase 3/9 activity was detected using a caspase 3/9 activity kit (Cat# K118-25; Beyotime Institute of Biotechnology) according to the manufacturer’s protocol. DEVD-p-NA substrate or LEHD-p-NA substrate (5 μL at 4 mM) at a final concentration of 200 μM were added to samples at 37°C for 1 or 2 hours. A wavelength of 405 nM was used to detect caspase 3/9 activity.
9
Caspase-3/9 Activity Assay in BMSCs
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BMSCs were washed with ice-cold phosphate buffered saline and treated with RIPA buffer (Beyotime Institute of Biotechnology) for 30 min at 4°C. Supernatants were centrifuged at 12,000 × g at 4°C for 10 min and collected to measure the supernatant proteins using the BCA method (Beyotime Institute of Biotechnology). Total protein (10 µg) was used to measure caspase-3/9 activity levels using caspase-3/9 activity kits (C1116 or C1158, Beyotime Institute of Biotechnology). The absorbance of the samples were measured at 405 nm with a spectrophotometric microplate reader (Bio-Rad 680; Bio-Rad Laboratories, Inc.).
10
Apoptosis Quantification in Endometrial Cells
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Endometrial stromal cells were lysed with lysis buffer (radioimmunoprecipitation assay buffer) containing a protease inhibitor cocktail (phenylmethanesulfonyl fluoride) and EDTA at 4°C for 30 min. Cells were centrifuged at 12,000 × g for 10 min at 4°C. Subsequently, the concentration of total protein was determined using a bicinchoninic acid (BCA) assay, and 10 µg/lane total protein was used to analyze caspase 3/9 activity levels using caspase 3/9 activity kits (C1115 and C1158, Beyotime Institute of Biotechnology). The absorbance was measured by spectrophotometry with a microplate reader (model 680; Bio-Rad Laboratories) at 405 nm.
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