Caspase-3 and caspase-9 activities were analyzed using the caspase-3 and caspase-9 activity assay kits (Beyotime) according to the manufacturer’s protocol. The cells were processed in the same way as 2.7 and were lysed by lysis buffer (50 μL) for 15 min on ice. Cell lysates were centrifuged at 12,000× g for 10 min at 4 °C, the supernatants were collected, and protein concentration was determined by the Bradford’s method. The reaction was added to the reaction buffer containing Ac-DEVD-pNA (10 μL) and Ac-LEHD-pNA (10 μL) and incubated for 2 h at 37 °C. Then, the samples were measured using the MicroplateReader (SPECTRAMAX 190, Molecular Devices, Sunnyvale, CA, USA) with a wavelength of 405 nm. The caspase-3 and caspase-9 activities were measured as a fold of enzyme activity in comparison with the control. All the experiments were conducted in triplicate.
Caspase 3 and caspase 9 activity assay kit
Manufactured by Beyotime
Sourced in China
The Caspase-3 and caspase-9 activity assay kit is a tool used to measure the enzymatic activity of caspase-3 and caspase-9 in cell lysates or purified protein samples. Caspase-3 and caspase-9 are important proteases involved in the apoptosis (programmed cell death) pathway. This kit provides a colorimetric or fluorometric method to quantify the activity of these caspases.
Automatically generated - may contain errors
Lab products found in correlation
Microplate reader, by Molecular Devices (1 mentions)
Spectramax id5, by Molecular Devices (1 mentions)
Lysis buffer, by Abcepta (1 mentions)
Thiazole blue, by Merck Group (1 mentions)
Dcfh2 da, by Merck Group (1 mentions)
Rhodamine 123, by Beyotime (1 mentions)
Dapi dye, by Beyotime (1 mentions)
Tnf α elisa kit, by Beyotime (1 mentions)
Annexin 5 fitc pi apoptosis kit, by BD (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Bde 47, by AccuStandard (1 mentions)
N acetyl cysteine nac, by Yuanye Bio-Technology (1 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Lysotracker red, by Thermo Fisher Scientific (1 mentions)
Mitotracker red, by Beyotime (1 mentions)
Reactive oxygen species assay kit, by Beyotime (1 mentions)
Atp assay kit, by Beyotime (1 mentions)
Mitochondrial membrane potential assay kit, by Beyotime (1 mentions)
Tissue mitochondria isolation kit, by Beyotime (1 mentions)
13 protocols using caspase 3 and caspase 9 activity assay kit
1
Caspase-3 and Caspase-9 Activity Assay
2
Caspase-3 and Caspase-9 Activity Assay
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The caspase‐3 and caspase‐9 activities were analyzed using caspase‐3 and caspase‐9 activity assay kits (Beyotime). Briefly, A549 and H1299 cells were lysed with lysis buffer. The reaction system was prepared according to the kit instructions. Cell absorbance was determined at 405 nm using a microplate reader (SpectraMax iD5; Molecular Devices), and relative activity was calculated according to standard curve.
3
Caspase Activation in TroBcl2-Modulated LPS Response
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To investigate the role of TroBcl2 on caspases activation during LPS stimulation, the activities of caspase 3 and caspase 9 were examined by the Caspase 3 and Caspase 9 Activity Assay Kits (Beyotime, Shanghai, China) according to the manuals. GPS cells were transfected with pTroBcl2 (or pCN3), and siTroBcl2 (or siTroBcl2-C) according to the transfection procedure described above. Then, 24 h after transfection, cells were stimulated by 2 μg/ml LPS. Non-transfected cells treated with LPS were as the control. Twenty-four hours after LPS stimulation, cells were digested and collected by centrifugation with 600 g at 4°C for 5 min. Then, the supernatant was removed and the precipitate cells was cleaned once with PBS. After that, cells were lysed with lysate buffer in ice bath for 15 min. Then cells were centrifuged at 20,000 g for 15 min at 4°C. The supernatant was used for detecting the caspase activity. The Ac-DEVD-pNA and Ac-LEHD-pNA were the substrates for caspase 3 and caspase 9, respectively. The absorbance at 405 nm was measured using a microplate reader of multi-wavelength (Thermo Fisher Scientifi, BioTek, USA). There were three independent replicates for the experiment.
4
Trophoblast Cell Apoptosis Assay
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Trophoblast cell apoptosis was evaluated using caspase-3 and caspase-9 activity assay kits (Beyotime). Briefly, the transfected trophoblast cell lysates were collected after the addition of lysis buffer (Abgent, Suzhou, Jiangsu, China). Cell lysates were treated with 2 mM Asp-Glu-Val-Asp (to assay caspase-3 activity) or 2 mM Leu-Glu-His-Asp (to assay caspase-9 activity), and labeled with p-nitroaniline for 2 h in the dark at 37°C. The OD value at 405 nm was measured using a Labserv K3 microplate reader (WoYuan, Shanghai, China).
5
BDE-47 Induced Apoptosis and Cytokine Response
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BDE-47 was purchased from AccuStandard (New Haven, CT, USA). DCFH2-DA, thiazole blue, and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO, USA). The Annexin V-FITC/PI apoptosis kit was purchased from BD Bioscience (Franklin Lake, NJ, USA). Arg-1 and iNOS antibodies were purchased from eBioscience (San Diego, CA, USA). E. coli (strain K-12) was purchased from Invitrogen (Carlsbad, CA, USA). The IL-10 enzyme-linked immunosorbent assay (ELISA) kit was purchased from MultiSciences (Hangzhou, China). Cytochrome C antibodies, IL-6, IL-1β, TNF-α ELISA kits, DAPI dye, rhodamine 123, and Caspase-3 and Caspase-9 activity assay kits were purchased from Beyotime Biotechnology (Shanghai, China). BAX, Bcl-2, and TNF-α antibodies were purchased from WanLeiBio Co., Ltd. (Shenyang, China). ECL luminescent reagent was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Real-time fluorescent quantitative reagents were purchased from Takara Biomedical Technology Co., Ltd. (Beijing, China). N-acetylcysteine (NAC) and L-buthionine-(S,R)-sulfoximine (BSO) were purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China).
6
Apoptosis Quantification by Flow Cytometry
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Cells were plated into six-well plates and digested with trypsin. After washing with PBS for three times, AnnexinV-fluorescein isothiocyanate (AnnexinV-FITC) and Propidium Iodide (PI) were added and incubated for 15 min. Then, the apoptotic rate was detected using an Attune NxT Flow Cytometer (Thermo Fisher Scientific, Waltham, MA, USA). In addition, the activities of caspase 3 and caspase 9 were examined using caspase 3 and caspase 9 activity assay kits (Beyotime, Shanghai, China) according to the manufacturer’s instructions.
7
Caspase Activity Assay for Hypoxia/Ischemia
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Caspase activities was measured by Caspase-3 and Caspase-9 Activity Assay Kit (Beyotime, Haimen, China). Briefly, NRCMs lysates were prepared after treatment with different dose of GS-Rb1 for 24 h under hypoxia/ischemia conditions. Assays were performed on 96-well microtitre plates by incubating 10 μL protein cell lysate per sample in 80 μL reaction buffer containing 10 μL substrate (Asp-Glu-Val-Asp (DEVD)-p-nitroaniline (pNA) for caspase-3, and Leu-Glu-His-Asp (LEHD)-pNA for caspase-9, 2 mM). Lysates were incubated at 37°C for 4–6 h. Samples were measured with a Microplate Reader (Bio-Rad, Hercules, CA) at an absorbance of 405 nm. Caspase activity was expressed as the percentage relative to the control group.
8
Caspase-3 and Caspase-9 Activity Assay
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Caspase-3 and caspase-9 activity was measured by using a caspase-3 and caspase-9 activity assay kit according to manufacturer's protocol (Beyotime Institute of Biotechnology, Chuzhou, China). At 48 h posttransfection, the OS cells (U-2OS and 143B) were harvested and lysed at 4°C for 30 min using lysis buffer. Total protein was incubated with 10 μL Ac-DEVD-pNA and 10 μL Ac-LEHD-pNA at 37°C for 2 h. The absorbance of each well was measured with a microplate reader set at 405 nm.
9
Mitochondrial Mechanisms of Camptothecin
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Camptothecin (CPT) (>98%) and 2-(dimethylamino) ethyl methacrylate (DEA) were acquired from Giant Medical Technology Co., Ltd. (Chengdu, China), whereas N-(3-aminopropyl) methacrylamide hydrochloride was purchased from Bide Pharmaceutical Technology Co., Ltd. (Shanghai, China). Lyso-tracker Red was provided by Thermo Fisher Scientific (Shanghai, China). MitoTracker Red, Mitochondrial Membrane Potential Assay Kit, Reactive Oxygen Species Assay Kit, ATP Assay Kit, Caspase 3 and Caspase 9 Activity Assay Kit, and Tissue Mitochondria Isolation Kit were all obtained from Beyotime Biotechnology Co., Ltd. (Shanghai, China). All other reagents were of analytical grade or above.
10
Caspase-3 and Caspase-9 Activity Assay
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Cells treated with or without 10 mg/L of GSPs for 24 h were lysed in cold lysis buffer and centrifuged at 14,000× g for 10 min at 4 °C. Caspase-3 and caspase-9 activity was determined using a commercial caspase-3 and caspase-9 activity assay kit (Beyotime Biotechnology, Shanghai, China). Briefly, 20 μL of cell lysate was mixed with 40 μL of caspase buffer and 10 μL of Ac-DEVD-pNA (2 mM) or Ac-LEHD-pNA (2 mM) and incubated at 37 °C for 4 h. The absorbance of the samples was then measured at a wavelength of 405 nm. The protein concentration of the samples was determined by the Bradford assay. The caspase-3 and caspase-9 activity contained in the unit weight protein was calculated based on total sample protein concentration.
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