Caspase 1 activity was assessed using a Caspase–1/ICE Fluorometric Assay kit (Biovision Inc, Milpitas, CA) based on the detection of cleavage substrate YVAD-AFC (AFC: 7-amino-4-trifluoromethyl coumarin). The cleavage product, free AFC emits a yellow-green fluorescence (λmax = 505nm). Briefly, adipose tissue was homogenized in chilled lysis buffer provided and 200 μg of total protein lysate was used in the assay. A standard curve was generated using human recombinant active caspase–1 (BioVision Inc.). Lysate or human recombinant active caspase–1 was incubated with reaction buffer (provided) and 50 μM of YVAD-AFC substrate for 2 hours at 37°C. The fluorescence signal was read using a fluorometer equipped with a 400 nm excitation filter and 505 nm emission filter.
Caspase 1 ice fluorometric assay kit
Manufactured by Abcam
Sourced in United States
The Caspase–1/ICE Fluorometric Assay kit is a laboratory equipment product that measures the activity of Caspase-1, also known as Interleukin-1β-converting enzyme (ICE). The kit utilizes a fluorogenic substrate that emits a fluorescent signal upon cleavage by active Caspase-1.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant human caspase 1, by Abcam (1 mentions)
Fmk001, by R&D Systems (1 mentions)
Synergy h1 hybrid multi mode reader, by Agilent Technologies (1 mentions)
Z vad fmk, by Apexbio (1 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (1 mentions)
Sc 203242, by Santa Cruz Biotechnology (1 mentions)
Microplate fluorometer, by Berthold Technologies (1 mentions)
4 protocols using caspase 1 ice fluorometric assay kit
1
Caspase-1 Activity Quantification
2
Caspase-1 Activity Inhibition Assay
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Recombinant human caspase-1 (1 unit/rx, #1081, BioVision Inc., CA. USA) was incubated with YVAD-pNA, a substrate of caspase-1, in the presence of lentinan or Z-VAD-FMK (FMK001, R&D Systems). Caspase-1 activity was measured using a Caspase-1/ICE Fluorometric Assay Kit (#K110, BioVision Inc.) according to the manufacturer’s protocol.
3
Mitochondrial Superoxide and Caspase-1 Assay
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BMDMs (1.25 × 105 cells per well) plated in a 96-well black plate (SPL Life Science Co.) were incubated with MitoSOXTM Red mitochondrial superoxide indicator (2.5 μM, M36008, Invitrogen) for 30 min at 37 °C. Cells were treated with rotenone (160 μM; sc-203242, Santa Cruz Biotechnology) in the presence of mercury, arsenic, or diphenyleneiodonium (DPI, 0504, 200 μM, Tocris Bioscience) or for 6 h at 37 °C. For caspase-1 activity, human recombinant caspase-1 (1 unit/rx, BioVision Inc., Milpitas, CA, USA) was incubated with obovatol or a caspase inhibitor (Z-VAD-FMK, 10 μg/ml; APExBIO, Boston, MA, USA) in the presence of caspase-1 substrate, YVAD-pNA, using a caspase-1/ICE Fluorometric Assay kit (BioVision Inc.). The plates were readout using a plate reader (510/580 nm, Synergy™ H1 Hybrid Multi-Mode Reader, BioTek).
4
Caspase-1 Activity Assay in AgNPs Exposure
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To monitor the activity of caspase-1, a caspase-1/ICE fluorometric assay kit (Biovision, USA) was used. This assay is based on the detection of the cleavage of the substrate YVAD-AFC, which emits blue light; after its cleavage by caspase-1, free AFC emits a yellow green fluorescence. According to the manufacturer's protocol, cells were exposed to TNFα (20 ng/ml) and AgNPs (10 or 100 µg/ml) separately and together. After 12 h of exposure, cells were pelleted, resuspended in cell lysis buffer, and incubated on ice for 10 min. Then, 2× reaction buffer and YVAD-AFC substrate were added to the cell lysate and incubated at 37°C for 1-2 h. Fluorescence was then measured using a fluorometric plate reader (Microplate Fluorometer, Twinkle LB 970, BERTHOLD TECHNOLOGIES) at an excitation/emission wavelength of 400 nm/505 nm. The fold increase in caspase-1 activity was determined by comparing the activity of the enzyme in the samples with the activity of the untreated cells.
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