Caspase-3, caspase-9, and caspase-12 activity was measured using respective caspase assay kits (Beyotime Biotechnology, China) 47 (link). Briefly, heart tissues were homogenized, centrifuged to obtain supernatants. Supernatants (100 µg protein) were loaded in 96-well plate, incubated with Ac-DEVD-pNA for 60 min at 37°C, and then quantified by microplate reader according to the manufacturer's instruction.
Caspase assay kit
Manufactured by Beyotime
Sourced in China
Caspase assay kits are laboratory tools used to measure the activity of caspase enzymes. Caspases are a family of proteases involved in various cellular processes, including apoptosis. These kits provide a quantitative method to assess caspase activity in biological samples.
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Lab products found in correlation
Softedge myocam system, by IonOptix (1 mentions)
Epoch 2, by Agilent Technologies (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Annexin 5 pi, by Vazyme (1 mentions)
Bradford protein assay kit, by Beyotime (1 mentions)
Microplate spectrophotometer, by Dynex (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Bradford method, by Beyotime (1 mentions)
10 protocols using caspase assay kit
1
Caspase Activity Assay in Heart
2
Assessing Cardiomyocyte Mechanics and Viability
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Mechanical properties of cardiomyocytes were assessed using a SoftEdge MyoCam system (IonOptix, Milton, MA) as our previously reported [27] (link). Cell shortening and relengthening were assessed using the following indices: resting cell length, peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90), and maximal velocity of shortening/relengthening (±dL/dt).
Cellular viability was detected via caspase3 and caspase9 activity as well as MMT assay. Caspase9 and caspase3 activities were determined with caspase assay kits (Beyotime, China), which detect the production of the chromophore p-nitroanilide after its cleavage from the peptide substrate DEVD-p-nitroanilide and LEHD- p-nitroanilide. The MMT assay was conducted as our previously reported [28] .
Cellular viability was detected via caspase3 and caspase9 activity as well as MMT assay. Caspase9 and caspase3 activities were determined with caspase assay kits (Beyotime, China), which detect the production of the chromophore p-nitroanilide after its cleavage from the peptide substrate DEVD-p-nitroanilide and LEHD- p-nitroanilide. The MMT assay was conducted as our previously reported [28] .
3
Evaluating Cell Viability via Caspase and MTT Assays
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Cell viability was detected by determining caspase-3 and -9 activity, as well as by MMT assay. Caspase-9 and -3 activities were determined using caspase-assay kits (Beyotime Institute of Biotechnology), which detect the production of the chromophore p-nitroanilide after its cleavage from the peptide substrate DEVD-p-nitroanilide and LEHD-p-nitroanilide (31 (link)). MMT assay was conducted as previously described (26 (link)). Briefly, the cells were seeded in a 96-well plate at a density of 1×106 cells in triplicate for 7 days at 37°C with 5% CO2. Subsequently, 20 μl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/ml; pH 7.4; Sigma-Aldrich) were added to the cells for 4 h. The supernatants were then discarded and 100 μl dimethyl sulfoxide (Sigma-Aldrich) was added to each well for 10 min. The OD of the samples was measured at an absorbance of 490 nm using a spectrophotometer (Epoch 2; BioTek Instruments, Inc., Winooski, VT, USA). The assay was repeated 3 times.
4
Apoptosis Induction in SVCV-Infected Cells
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Cells seeded in 6-well culture plates were infected with 100TCID50 SVCV for 2 h, after that the medium was replaced with new maintenance medium containing 1.6 mg/L ARG and incubated for 48 and 72 h. Apoptosis assay was performed using Annexin V/PI (Vazyme, China) staining (Zhu et al., 2016 (link)). The activities of caspase-3, -8 and -9 was measured using caspase assay kits (Beyotime, China) (Zhu et al., 2015 (link)).
5
Caspase Activity Assay Protocol
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The activities of caspase-3 and caspase-9 were analyzed with corresponding Caspase Assay Kits (Beyotime or Solarbio, China). Briefly, proteins were extracted from cells and then qualified with Bradford Protein Assay Kit (Beyotime, China). Subsequently, samples were incubated with the caspase substrate for 24 h at 37 °C. The absorbance was determined at 405 nm.
6
Caspase Enzymatic Activity Assay
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Caspase 3, caspase 9 and caspase 8 activities were determined by spectrophotometric method (Caspase Assay kit, Beyotime, Jiangsu, China). Treated cells were harvested, suspended in cell lysis buffer (80–100 µl) and incubated on ice for 30 min, after centrifugation at 10,000 × g for 15 min at 4°C, supernatants were collected and placed in 96-well plates and then 10 µl specific caspase substrates (Ac-DEVD-pNA) was added. Plates were incubated at 37°C for 2 h and caspase activities were tested by spectrophotometer (Spectrum, Shanghai, China).
7
Caspase-3 Activity Assay Protocol
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Caspase-3 activity was detected with a caspase assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. In brief, 2×106 cells were lysed with 100 µl lysis buffer on ice for 15 min and the cell lysates were then centrifuged at 16,000 × g for 10 min at 4°C. Reaction buffer (80 µl) and acetyl-DEVD-p-nitroanilin (pNA; 10 µl) was mixed with 10 µl lysate and the mixture was then incubated for 2 h at 37°C. The optical density of free pNA was measured at an absorption wavelength of 405 nm using a microplate spectrophotometer (Dynex Technologies, Chantilly, VA, USA). Caspase-3 activity was expressed as the relative absorbance ratio of samples vs. control group.
8
Caspase-3 Activity Measurement in NRCs
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To measure caspase-3 enzymatic activity, the NRCs were cultured for 48 h followed by treatment with P-1075 (100 µM), HMR-1098 (30 µM) and/or RR (50 µM) for 30 min following treatment with or without LPS (25 µg/ml). The activity of caspase-3 was determined using a caspase assay kit (Beyotime Institute of Biotechnology) based on the ability of caspase-3 to change acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) into a yellow formazan product p-nitroaniline (pNA). Protein concentrations were determined using a Bradford protein assay (Bio-Rad, Hercules, CA, USA).
9
Caspase Activity Measurement in K562 Cells
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Caspase-3 and caspase-9 activity were analyzed with a caspase assay kit (Beyotime). The kit is based on the spectrophotometric detection of the chromophore p-nitroanilide (pNA) after cleavage from the substrates Ac-DEVD-pNA and Ac-LEHD-pNA. After incubation with chaetominine, K562 cells were collected and washed with PBS. Cells were suspended in lysis buffer and incubated on ice. After centrifugation, the supernatant was incubated with the substrates. Substrate hydrolysis was monitored at 405 nm with a microplate reader (SpectraMax® i3, Austria), and the fold increase in caspase activity was determined by comparing the results of chaetomi-nine-treated samples to that of the untreated controls.
10
Caspase Activity Determination Protocol
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Caspase-9, caspase-8, and caspase-3 activities were determined using a caspase assay kit according to the manufacturer’s instructions (Beyotime, China). Briefly, cells were lysed and protein concentrations were determined by the Bradford method (Beyotime, China). Caspase-3, caspase-8 and caspase-9 activities were measured using their respective substrate peptides Ac-DEVD-ρNA, Ac-IETD-ρNA and Ac-LEHD-ρNA. A mixture of 80 μl of detection buffer, 10 μl of sample and 10 μl of either Ac-DEVD-ρNA, Ac-IETD-ρNA or Ac-LEHD-ρNA was incubated at 37°C for 6 h. Release of ρ-nitroanilide (ρNA) was quantitated by measuring absorbance at 405 nm with a microplate reader. Caspase-3, caspase-8 and caspase-9 activities were calculated based on a standard curve. Blank values were subtracted, and increases in caspase-3 activities were expressed as fold increase and calculated based on activities measured from the control group. All experiments were performed in triplicate.
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