The DEVDase analysis for apoptosis was used. Briefly, 24 hours after irradiation, cells were lysed with a 0.5% NP40 lysis buffer, and 50 μL of lysate was incubated with 10 mmol/L Ac-DEVD-AMC substrate (Sigma) at 37°C for 1, 2, 3 and 4 hours. Substrate fluorescence was detected using a SpectraMax Gemini (Molecular Probes, Carlsbad, CA) at 380-nm excitation, 460 nm emission, and normalized to protein concentration.
Ac devd amc substrate
Manufactured by Merck Group
Sourced in United States
Ac-DEVD-AMC substrate is a fluorogenic peptide substrate used to detect and measure caspase-3 activity in biological samples. It consists of the amino acid sequence Ac-Asp-Glu-Val-Asp-AMC, where AMC is the fluorogenic reporter molecule. Upon cleavage by caspase-3, the AMC moiety is released, resulting in an increase in fluorescent signal that can be quantified.
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Lab products found in correlation
4 protocols using ac devd amc substrate
1
DEVD-based Apoptosis Quantification
2
Caspase-3 Activity Assay
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Cells were lysed with a 0.5% NP40 lysis buffer, and 50 μg of protein lysates were incubated with 10 mmol/L Ac-DEVD-AMC substrate (Sigma) at 37 °C for 2 h. Fluorescence was detected using a SpectraMax Gemini (Molecular Probes, Carlsbad, CA, USA): 360-nm excitation and 460 nm emission.
3
Caspase 3 Activity Quantification Protocol
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Caspase 3 activity was analyzed as described previously [25 (link)]. Isolated cells were washed twice with PBS (5 min, 250 g, 4°C) and lysed (50 mM HEPES; 5 mM CHAPS; 5 mM DTT) on ice for 20 min and centrifuged (15 min, 15 000 g, 4°C). Lavage fluid was diluted 1 : 1 with 2x concentrated lysing buffer. The proteins present in supernatants were quantified using Coomassie® Protein Assay (Bio-Rad, USA) and diluted to an equal concentration. 5 μg of protein samples was incubated in an assay buffer in parallels (20 mM HEPES; 2.5 mM CHAPS, 5 mM DTT, 2 mM EDTA) containing 50 μM of caspase 3 (Ac-DEVD-AMC) substrate (Sigma-Aldrich, USA) at 37°C for 4 h. The level of fluorescence was determined using microplate reader (Infinite 200, Tecan, Switzerland; 360 nm excitation, 460 nm emission).
4
Caspase-3 Activity Quantification
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As we previously described [41 (link)], cells were lysed with a 0.5% NP40 lysis buffer, and 50 μg of protein lysates was incubated with 10 mmol/L Ac-DEVD-AMC substrate (Sigma) at 37 °C for 2 h. Fluorescence was detected using a SpectraMax Gemini (Molecular Probes, Carlsbad, CA) with 360 nm excitation and 460 nm emission.
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