Neuroblastoma cells (106 cells/mL) were seeded on 6-wells plates 24 h prior the pretreatment with wine constituents (0.1–10 μM; 18 h) or MAPK inhibitors (0.5–50 μM; 1 h) exposure to SIN-1 (1 mM; 6 h). Culture was then washed twice with ice-cold PBS + EGTA + EDTA (200 μM), and the cells were lysed and collected as previously described. The activity of caspase-3-like proteases in the lysates was determined using the caspase-3 colorimetric assay kit (Sigma, Poole, Dorset, UK) according to the manufacturer’s protocol, with the exception that 30 μL of cell lysate was used in assays. Absorbance data (405 nm) obtained using the caspase-3 inhibitors were subtracted from the absorbance obtained without caspase-3 inhibitor to correct for any non-specific hydrolysis. Vehicle controls and blanks were incorporated, and caspase-3 protein was used as a positive control of the assay. Assays were carried out in triplicates.
Caspase 3 colorimetric assay kit
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany
The Caspase-3 colorimetric assay kit is a laboratory tool used to detect and measure the activity of the enzyme caspase-3. Caspase-3 is a key mediator of apoptosis, or programmed cell death. The kit provides a method for quantifying caspase-3 activity in cell lysates or other biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Spectramax 190 microplate reader, by Molecular Devices (3 mentions)
Elia kits, by Cusabio (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Microplate reader, by Agilent Technologies (2 mentions)
Brdu cell proliferation chemiluminescent assay kit, by Cell Signaling Technology (1 mentions)
Caspase 8 colorimetric assay kit, by Abcam (1 mentions)
Flexstation 3 microplate reader, by Molecular Devices (1 mentions)
Varioskan lux multimode microplate reader, by Thermo Fisher Scientific (1 mentions)
Phloridzin, by Merck Group (1 mentions)
Phloretin, by Merck Group (1 mentions)
Dcfda cellular reactive oxygen species detection assay kit, by Abcam (1 mentions)
Cytotox 96 non radioactive cytotoxicity assay ldh, by Promega (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay mts, by Promega (1 mentions)
α linolenic acid, by Merck Group (1 mentions)
Stearic acid, by Merck Group (1 mentions)
Oleic acid, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Linoleic acid, by Merck Group (1 mentions)
Ac devd pna, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
55 protocols using caspase 3 colorimetric assay kit
1
Caspase-3 Assay in Neuroblastoma Cells
2
Cell Proliferation and Apoptosis Assays
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Cell Proliferation was measured using a BrdU cell proliferation chemiluminescent assay kit (Cell Signaling Technologies, New England Biolabs, Frankfurt am Main, Germany). Apoptosis was determined using the Caspase-3 colorimetric assay kit (Sigma Aldrich GmbH, Steinheim, Germany). Both assays were performed according to the manufacturer’s protocols.
3
Caspase Activity Quantification in HepG2 Cells
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A caspase-3 colorimetric assay kit (Sigma-Aldrich), caspase-8 colorimetric assay kit (BioVision Inc.; Milpitas, CA, USA) and caspase-9 colorimetric assay kit (Invitrogen; Frederick, MD, USA) were used to investigate the activation of caspase-3, -8 and -9 in RCX-treated HepG2, respectively. The analysis was performed according to the manufacturer’s instructions. Briefly, cells were lysed by incubation with cell lysis buffer on ice for 10 min and then centrifuged. Enzyme reactions were carried out in a 96-well flat-bottom microplate. To each reaction mixture, 5 μL of cell lysate was added. Absorbance at 405 nm was measured using the SpectraMax 190 Microplate Reader (Molecular Devices). The results are expressed as the specific activity (IU/mg protein) of each caspase.
4
Quantifying Caspase-3 Activity in HCT116 Cells
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A caspase-3 colorimetric assay kit (Sigma-Aldrich Co., Saint Louis, MO, USA) was used to investigate caspase-3 activation on complexes-treated HCT116 cells based on the cleavage of DEVD-pNA and the analysis was performed according to the manufacturer’s instructions. Briefly, cells were lysed by incubation with cell lysis buffer on ice for 10 min and then centrifuged. Enzyme reactions were carried out in a 96-well flat-bottom microplate. To each reaction mixture, 5 μL cell lysate was added. Absorbance at 405 nm was measured using the SpectraMax 190 Microplate Reader (Molecular Devices, Sunnyvale, CA, EUA). The results were expressed as specific activity (IU/mg protein) of caspase-3.
5
Caspase-3 Activity in Neutrophils
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Neutrophils (1 × 106/ml) were cultured for 18 h at 37 or 39.5 °C in 5% CO2 at pH values of 7.3, 6.5, and 6.0. Cells were then harvested and processed according to the caspase 3 colorimetric assay kit (Sigma-Aldrich). Absorbance due to the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) was measured at 405 nm using the FlexStation 3 microplate reader (Molecular Devices Inc., Sunnyvale, CA, USA), and caspase 3 activity was expressed as OD405 values.
6
Measuring Caspase-3 Activity in Aphid Gut Tissues
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Dissected gut tissues of
R. padiadults were incubated in ice-cold lysis buffer (50 mM HEPES, pH 7.4, 5 mM CHAPS, and 5 mM DTT). The caspase 3 activity assay was carried out with the application of Caspase 3 Colorimetric Assay Kit (Sigma–Aldrich, Poznań, Poland, PC CASP-3-C), following the manufacturer’s protocol. This assay is based on the amount of
p-nitroaniline released from hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp-pnitroanilide (Ac-DEVD-pNA) by caspase 3. The assay can be performed in 1 cm
3volume and measured using a spectrophotometer. Activity of caspase-3 was expressed as nanomoles (nmol) of released
p-nitroaniline per minute per cm
3. Three insect sampling were made for each assay.
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Dissected gut tissues of
R. padiadults were incubated in ice-cold lysis buffer (50 mM HEPES, pH 7.4, 5 mM CHAPS, and 5 mM DTT). The caspase 3 activity assay was carried out with the application of Caspase 3 Colorimetric Assay Kit (Sigma–Aldrich, Poznań, Poland, PC CASP-3-C), following the manufacturer’s protocol. This assay is based on the amount of
p-nitroaniline released from hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp-pnitroanilide (Ac-DEVD-pNA) by caspase 3. The assay can be performed in 1 cm
3volume and measured using a spectrophotometer. Activity of caspase-3 was expressed as nanomoles (nmol) of released
p-nitroaniline per minute per cm
3. Three insect sampling were made for each assay.
7
Caspase-3 Activity Colorimetric Assay
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Caspase-3 activity was assessed with a colorimetric assay based on the hydrolysis of acetyl–Asp–Glu–Val–Asp p-nitroanilide (Ac-DEVD-pNA) by Caspase-3 and releasing p-nitroaniline (pNA). MDA-MB-231 cell lysates (100 ng/mL) were diluted and subjected to analysis via Caspase-3 Colorimetric Assay Kit (Sigma-Aldrich, Saint Louis, MO, USA) based on the protocol of the manufacturer. The reaction for color development was held at 37 °C for 2 h, and the value of OD405 was determined using a Varioskan TM LUX multimode microplate reader (Thermo Scientific, Waltham, MA, USA). Each caspase-3 activity was expressed as a value of OD405.
8
Synthesis and Evaluation of Phloridzin Esters
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Fatty acid esters of Phloridzin (Pz) viz. stearic acid ester of Pz (Pz-stearic acid), oleic acid ester of Pz (Pz-oleic acid), linoleic acid ester of Pz (Pz-linoleic acid), α-linolenic acid ester of Pz (Pz-α-linolenic acid), DHA ester of Pz (Pz-DHA) and eicosapentaenoic acid ester (EPA) of Pz (Pz-EPA) were synthesised in our laboratory as previously reported [17] (link).
Phloridzin, phloretin, caspase 3 colorimetric assay kit, propidium iodide, fatty acids namely oleic acid, stearic acid, linoleic acid, α-linolenic acid, EPA and DHA were purchased from Sigma-Aldrich Canada. Cell Titer 96 Aqueous One solution cell proliferation (MTS) assay, CytoTox 96 non-radioactive cytotoxicity (LDH) assay and CellTiter-Glo luminescent assay kits were purchased from Promega, Madison, WI, USA. Sterile dimethyl sulfoxide (DMSO) (ATCC), GFP-certified apoptosis/necrosis detection kit for microscopy from Enzo Life Sciences, Brockville, ON, Canada; ApoTarget Quick Apoptotic DNA Ladder Detection kit from Invitrogen, Burlington, ON, Canada; DCFDA-Cellular Reactive Oxygen Species detection assay kit form Abcam, Toronto, ON, Canada; and 5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) from Cayman Chemicals, Burlington, ON, Canada were also used for the study.
Phloridzin, phloretin, caspase 3 colorimetric assay kit, propidium iodide, fatty acids namely oleic acid, stearic acid, linoleic acid, α-linolenic acid, EPA and DHA were purchased from Sigma-Aldrich Canada. Cell Titer 96 Aqueous One solution cell proliferation (MTS) assay, CytoTox 96 non-radioactive cytotoxicity (LDH) assay and CellTiter-Glo luminescent assay kits were purchased from Promega, Madison, WI, USA. Sterile dimethyl sulfoxide (DMSO) (ATCC), GFP-certified apoptosis/necrosis detection kit for microscopy from Enzo Life Sciences, Brockville, ON, Canada; ApoTarget Quick Apoptotic DNA Ladder Detection kit from Invitrogen, Burlington, ON, Canada; DCFDA-Cellular Reactive Oxygen Species detection assay kit form Abcam, Toronto, ON, Canada; and 5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) from Cayman Chemicals, Burlington, ON, Canada were also used for the study.
9
Caspase-3 Activity Assay in Lung Tissue
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Caspase-3 protease activity in the lung tissue was determined using a caspase-3 colorimetric assay kit (Sigma-Aldrich; Merck KGaA), in accordance with the manufacturer's protocol. In brief, following homogenization of the whole lung tissue in cell lysis buffer, homogenates were centrifuged for 1 min at 10,000 × g, and the supernatant was extracted and incubated with Asp-Glu-Val-Asp-p-nitroanilide (pNA) and reaction buffer for 90 min at 37°C. Levels of the chromophore pNA were quantified using a spectrophotometer at 405 nm, which reflected the caspase-3 activity. The data were normalized to the lung weight.
10
Caspase-3 Activity Assay in Liver Tissue
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Caspase-3 activity was analyzed in liver specimen lysate using the caspase-3 colorimetric assay kit (Sigma-Aldrich, MI, USA; #CASP-3-C), adopting the suppliers’ instructions.
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