Cells were lysed (#7018; Cell Signaling) and, 5-10 μg of the lysate was mixed with 150 μl protease assay buffer containing 20 mM HEPES (pH 7.5), 10% glycerol, and 2 mM DTT, and 20 μM fluorogenic substrate Ac-DEVD-AMC (#556449, BD Pharmingen), followed by a 2-hour incubation at 37°C in a black 96-well plate. Fluorescence intensity was determined (excitation, 360 nm; emission, 460 nm) with a FLUOstar Omega plate reader (BMG labtech, Germany).
Ac devd amc
Manufactured by BD
Sourced in United States
Ac-DEVD-AMC is a fluorogenic substrate used for the detection and measurement of caspase-3 activity. It contains the amino acid sequence Ac-Asp-Glu-Val-Asp, which is the recognition sequence for caspase-3. When Ac-DEVD-AMC is cleaved by active caspase-3, the 7-amino-4-methylcoumarin (AMC) fluorophore is released, resulting in a measurable fluorescent signal.
Automatically generated - may contain errors
Lab products found in correlation
Ac ietd amc, by BD (2 mentions)
Versafluor fluorometer, by Bio-Rad (2 mentions)
Ac devd cho, by BD (2 mentions)
Fluostar optima microplate reader, by BMG Labtech (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
Staurosporine, by Merck Group (1 mentions)
Ls 50b luminescence spectrophotometer, by PerkinElmer (1 mentions)
Free amc, by Merck Group (1 mentions)
Chaps, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Apoalert dna fragmentation assay kit, by Takara Bio (1 mentions)
Mitotracker red cmxros dye, by Thermo Fisher Scientific (1 mentions)
N acetyl l cysteine, by Merck Group (1 mentions)
Aldetect lipid peroxidation assay kit, by Enzo Life Sciences (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Cytofluor series 4000, by Thermo Fisher Scientific (1 mentions)
32 protocols using ac devd amc
1
Caspase-3 Activity Assay
2
Fluorometric Caspase-3 Activity Assay
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Fluorometric assays of caspase activity were carried out by using the substrate Ac-DEVD-AMC (BD Pharmingen, San Diego, CA, USA) for caspase-3. Both A2780 and A2780CR cells were seeded at 1 × 104 cells/well in costar 96-well black plates and incubated at 37 °C and 5% CO2 in a humidified atmosphere for 24 h. Then, the cells were treated for 6 and 24 h with different concentrations of melittin to measure caspase-3 activity. Staurosporine (Sigma-Aldrich, Dorset, UK) was used to induce apoptosis at a concentration of 10 μM. The control cells were treated with media alone. The caspase-3 assay buffer was prepared as described previously [55 (link)]. The caspase-3 assay buffer (3×) was added to each well and incubated at 37 °C in 5% CO2 for 1 h. Fluorescence was measured at 360 nm (excitation) and 460 nm (emission) using a Spectramax M3 microplate reader. The average fluorescence values of the background were subtracted from the fluorescence values of experimental wells. Statistical analysis was done using one-way ANOVA followed by Bonferroni’s Multiple Comparison test.
3
Fluorometric Assay of Caspase Activity
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Fluorometric assays of caspase activity were carried out by using the substrate Ac-DEVD-AMC (BD Pharmingen, San Diego, CA) for caspase 3 and Ac-IETD-AMC (BD Pharmingen, San Diego, CA) for caspase 8. Briefly, cells were lysed in lysis buffer (10 mM HEPES, 142 mM KCl, 5 mM MgCl2, 1 mM EDTA, 0.2% NP-40 and pH 7.2) with 10 mM DTT. Following incubation for 30 min on ice, samples were centrifuged at 12,000 rpm for 30 min at 4°C and the protein content in supernatants was determined by Bradford dye method. Aliquots of 10 mg/100 mL assay volume were incubated with 140 mM site-specific tetrapeptide substrates Ac-DEVD-AMC for caspase 3 and Ac-IETD-AMC for caspase 8 in a caspase assay buffer (20 mM HEPES, 100 mM NaCl, 1 mM EDTA, 0.01% (w/v) CHAPS, 10% (w/v) sucrose and pH 7.2) with 10 mM DTT for 30 min. The release of the fluorogenic group AMC was determined at 37°C in a VersaFluor Fluorometer (Bio-Rad, Hercules, CA) with excitation at 380 nm and emission at 440 nm.
4
Caspase-3 Activity Assay in Liver
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Portions of frozen livers were processed and then assayed for specific caspase activities following manufacturers’ instructions using commercially available caspase-3 fluorogenic substrate, Ac-DEVD-AMC (BD Biosciences, San Diego, CA, USA). Caspase activity was evaluated by measuring the release of AMC (7-Amino-4-methylcoumarin) obtained by the cleavage of the defined synthetic peptide sequence by caspase-3, using a Perkin Elmer Luminescence Spectrophotometer LS 50B. Free AMC obtained from Sigma-Aldrich (St Louis, MO, USA) was used as the standard. Protein was determined using the BCA Protein Assay kit from Pierce. The caspase-3 activity was expressed as pmoles of AMC produced per mg liver protein.
5
Caspase-3 Activity Measurement Protocol
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After RN isolation, tissues were homogenized in lysis assay buffer containing 100 mM Hepes (Sigma, H4034), pH 7.4, 0.1% Chaps (Sigma, 3023), 1 mM EDTA (Sigma, E5134), 10 mM DTT (Sigma, D9779), and 1 mM PMSF (Sigma, 7626), and lysed by subsequent freezing in liquid N2 and thawing at 37°C three times. After centrifugation at 11 500 × g for 5 min, the protein concentration of resulting supernatant was quantified, and proteins were incubated at 37°C in lysis assay buffer containing 50 mM Ac-DEVD-AMC (BD PharMingen, San Jose, CA, USA; 556449). The fluorescence was measured at an excitation and emission wavelength of 380 and 460 nm, respectively, as previously described.54 (link) Caspase-3 activity in the SCH animals was expressed as percent increase in absorbance relative to CTRL animals.
6
Caspase-3 Activation Assay
7
Caspase 3 Activity Quantification in Liver
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Caspase 3 activity was quantified using the caspase 3 synthetic fluorogenic tetrapeptide substrate Ac-DEVD-AMC (BD Pharmingen) as previously we reported [12 (link)] using fresh liver tissue, values were normalized regarding Chow sample and reported as fold change.
8
Apoptosis-Inducing Agents and Assays
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DETA-NONOate was purchased from Cayman Biochemicals (Ann Arbor, MI), Calpain Inhibitor III was from Calbiochem (Darmstadt, Germany), and N-acetyl-l-cysteine (NAC) was purchased from Sigma Aldrich (St. Louis, MO). Ac-DEVD-AMC was purchased from Pharmingen (San Diego, CA, USA). ApoAlert DNA Fragmentation Assay kit was obtained from Clontech (Mountain View, CA). SOD activity was measured by using an assay kit obtained from Cayman Chemical Company (Ann Arbor, MI). Aldetect Lipid Peroxidation Assay Kit was from Enzo Life Sciences (Ann Arbor, MI, USA). MitoTracker Red CMX-Ros dye was obtained from Life Technologies (Grand Island, NY).
9
Caspase-3 Activity Assay
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Cell lysates (50 µg) were incubated with 200 nM Ac-DEVD-AMC (BD Biosciences, USA) in reaction buffer (20 mM HEPES, pH 7.5, 2 mM DTT, and 10% glycerol) at 37 °C. Per the manufacturer’s protocol, the reaction was monitored by fluorescence emission at 430 nm (excitation at 380 nm).
10
Gpr158 Expression Regulates Apoptosis
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BTSCs or GBM primary cells with different expression levels of Gpr158 (or GPR158) were lysed using lysis buffer (#7018; Cell Signaling). Subsequently, 40 μg of the samples was diluted to a final volume of 150 μl with protease assay buffer (20 mM HEPES (pH 7.5), 10% glycerol, and 2 mM DTT), and supplemented with 20 μM caspase-3 preferred, fluorogenic substrate Ac-DEVD-AMC (#556449, BD Pharmingen) for a 2-hour incubation 37 °C in a 96-well plate as per the manufacturer’s instruction. Fluorescence was determined (excitation, 360 nm; emission, 460 nm) with a CytoFluor series 4000 plate reader (Applied Biosystems). Background fluorescence was determined in wells containing the assay buffer only.
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