Caspase 3 Assay Kit Fluorimetric (Sigma-Aldrich) was used to assess the induction of apoptosis. HeLa cells (15,000 cells per well) were incubated for 2 h with Compound 21 (0.4 µM) in culture 96-well plates at 37 °C and 5% CO2. Then exposed to red light (λ = 660 nm, 20 min) and incubated for 40 min and 21 h 40 min. Analogous conditions but without exposure to light were used in the control. After incubation, the medium was removed, cells were washed with 200 µL of PBS, and 25 µL of lysis solution from Caspase 3 Assay Kit Fluorometric (Sigma-Aldrich) was added. All subsequent procedures were performed according to the manufacturer’s protocol. Fluorescence was measured on “Fluorat-02 Panorama” (Lumex). The difference between the values obtained for the wells with and without specific Caspase 3 inhibitor was calculated to estimate a degree of the specific substrate digestion. Data were normalized according to the DNA concentrations in the corresponding lysates measured with PicoGreen® (Molecular Probes, Eugene, OR, USA). The results were presented in pmol of the digested labeled substrate per amount of sample containing 1 ng of DNA based on the calibration ladder built using the clear fluorescent label, 7-amido-4-methylcoumarin.
Caspase 3 fluorimetric assay kit
Manufactured by Merck Group
Sourced in United States
The Caspase 3 Fluorimetric Assay Kit is a laboratory instrument designed to measure the activity of the caspase-3 enzyme. Caspase-3 is a key player in the apoptosis (programmed cell death) pathway. The kit utilizes a fluorogenic substrate specific for caspase-3, allowing for the quantitative determination of caspase-3 activity in cellular or tissue extracts.
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Microplate reader, by Agilent Technologies (1 mentions)
Bicinchoninic acid protein assay, by Thermo Fisher Scientific (1 mentions)
Valinomycin, by Merck Group (1 mentions)
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Hbss solution, by Merck Group (1 mentions)
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13 protocols using caspase 3 fluorimetric assay kit
1
Caspase 3 Assay of Photosensitizer
2
Fluorometric Caspase-3 Activity Assay
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Caspases‐3 activity was assessed using a Caspase‐3 Assay Kit, Fluorimetric (SIGMA) as described previously (Yamamoto et al., 2011 ). Briefly, cells were seeded into 24‐well plates at a density of 2.5 × 105 cells/cm2 followed by 24 hr incubation. After exposure to drugs for 24 hr, the supernatants were aspirated and cells were harvested with lysis buffer (50 mM HEPES, pH 7.4, 5 mM CHAPS and 5 mM DTT). The reaction buffer, including Acetyl‐Asp‐Glu‐Val‐Asp‐7‐amido‐4‐methylcoumarin (Ac‐DEVD‐AMC) and caspase‐3 specific substrates, was added to wells, and the production of AMC was sequentially detected in a CytoFluor® Plate Reader at an excitation wavelength of 360 nm/emission 460 nm. Enzyme activities were determined as initial velocities expressed as nmol AMC min−1 ml−1. They were then corrected with the quantity of protein in each well detected by BCA protein assays (Thermo Fisher Scientific).
3
Assessing Caspase-3 Activity in Endothelial Cells
4
Fluorimetric Caspase-3 Activity Assay
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A fluorimetric caspase 3 assay kit (Sigma-Aldrich) was used following the manufacture’s protocol. This assay is based on the hydrolysis of the peptide substrate Ac-DEVD-AMC (acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin) by caspase-3, which results in the release of fluorescent 7-amino-4-methylcoumarin (AMC). In brief, cells were lysed with 50 mM HEPES, 5 mM CHAPS, 5 mM DTT, pH 7.4 for 20 min on ice, and the assay buffer containing the Ac-DEVD-AMC substrate (20 mM HEPES, 2 mM EDTA, 0.1% CHAPS, 5 mM DTT, 16 µM Ac-DEVD-AMC, pH 7.4) was added. Aliquots of 200 µl were transferred to a 96-wells plate and the fluorescence recorded for 30 mins at 5 mins intervals at 37 °C (λexc = 360 nm, λem = 460 nm). CSP-3 activity was determined as AMC release rate extrapolating the slopes to those obtained from an AMC standard curve. Results are expressed as fold change, arbitrarily assigning the value of 1 to control cells.
5
Fluorimetric Caspase-3 Activity Assay
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A fluorimetric caspase-3 assay kit (Sigma-Aldrich) was used following the manufacturer’s protocol. Cells were lysed with 50 mM Hepes, 5 mM CHAPS, and 5 mM dithiothreitol (DTT) (pH 7.4) for 20 min on ice, and the assay buffer containing the Ac-DEVD-AMC (acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin) substrate [20 mM Hepes, 2 mM EDTA, 0.1% CHAPS, 5 mM DTT, and 16 μM Ac-DEVD-AMC, (pH 7.4)] was added. Aliquots of 200 μl were transferred to a 96-well plate, and the fluorescence was recorded at 5-min intervals for 30 min at 37°C using a Fluoroskan Ascent FL (Thermo Fisher Scientific) fluorimeter (excitation, 360 nm; emission, 460 nm). Caspase-3 activity was determined as AMC release rate extrapolating the slopes to those obtained from the AMC standard curve. Results were expressed as picomol per hour per microgram protein.
6
Cisplatin-Rapamycin Cytotoxicity Assay
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Cortical neurons were incubated in culture medium containing 10 μM Cisplatin ± 100 nM Rapamycin for 24 h. Control neurons were incubated in the absence of treatments. The toxicity of cisplatin-rapamycin co-treatment was evaluated by the activity of caspase-3 (Fluorimetric Caspase-3 Assay Kit, Sigma-Aldrich), according to the manufacturer’s instructions. This assay is based on the hydrolysis of the peptide substrate Ac-DEVD-AMC (acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin) by caspase-3, which results in the release of fluorescent 7-amino-4-methylcoumarin (AMC). Caspase-3 activity was determined as AMC release rate extrapolating the slopes to those obtained from an AMC standard curve. Results are expressed as mean activity ± SEM from 5 different neuronal cultures.
7
Caspase 3 Activity Measurement in DCX-SLN Treated Cells
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Caspase
3 activity was determined
using a Sigma-Aldrich Caspase 3 Fluorimetric Assay Kit. In brief,
TC-1 cells were seeded in 24-well plates at 25,000 cells/well and
incubated overnight. The cells were treated with DCX-SLNs (prepared
with trimyristin), DCX in T80/E, blank SLNs, or T80/E for 72 h. The
concentration of the DCX was 0.01 μM. The cells were then washed
with PBS and lysed. The cell lysate was centrifuged at 18000g for 10 min at 4 °C. The supernatant was transferred
to a clear-bottomed black plate and mixed with the assay substrate,
acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcourmarin (Ac-DEVD-AMC). The
mixture was incubated for 6 h for the hydrolysis of the Ac-DEVD-AMC
by caspase 3 to release the fluorescent AMC, which was quantified
by measuring the fluorescence intensity at 360 nm (excitation)/460
nm (emission) according to the manufacturer’s instruction.
The unit of the caspase 3 activity was mol AMC/min/mL. A caspase 3
inhibitor (provided in the kit) was used to confirm that the fluorescence
was due to caspase 3 activity. Total protein concentration in the
cell lysates was determined using Bio-Rad DC Protein Assay Kit following
the manufacturer’s instruction.
3 activity was determined
using a Sigma-Aldrich Caspase 3 Fluorimetric Assay Kit. In brief,
TC-1 cells were seeded in 24-well plates at 25,000 cells/well and
incubated overnight. The cells were treated with DCX-SLNs (prepared
with trimyristin), DCX in T80/E, blank SLNs, or T80/E for 72 h. The
concentration of the DCX was 0.01 μM. The cells were then washed
with PBS and lysed. The cell lysate was centrifuged at 18000g for 10 min at 4 °C. The supernatant was transferred
to a clear-bottomed black plate and mixed with the assay substrate,
acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcourmarin (Ac-DEVD-AMC). The
mixture was incubated for 6 h for the hydrolysis of the Ac-DEVD-AMC
by caspase 3 to release the fluorescent AMC, which was quantified
by measuring the fluorescence intensity at 360 nm (excitation)/460
nm (emission) according to the manufacturer’s instruction.
The unit of the caspase 3 activity was mol AMC/min/mL. A caspase 3
inhibitor (provided in the kit) was used to confirm that the fluorescence
was due to caspase 3 activity. Total protein concentration in the
cell lysates was determined using Bio-Rad DC Protein Assay Kit following
the manufacturer’s instruction.
8
Quantitative Caspase-3 Activity Assay
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Caspase-3 activity was determined by the Caspase-3 Fluorimetric assay kit (Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. Briefly, the cells were lysed with lysis buffer and incubated at 4°C for 10 min. The lysate was centrifuged at 16,000 × g for 5 min at room temperature. The supernatant was removed and incubated with an equal volume of assay buffer containing substrate (Ac-DEVD-AMC) at 37°C for 2 h. The absorbance of samples was measured at 405 nm using a BioTek microplate reader (Agilent Technologies, Inc.). The caspase-3 activity of each formulation was compared with that of the control group. The experiments were performed in triplicate.
9
Caspase-3 Activity Quantification
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Casp-3 activity was measured using a Caspase 3 Fluorimetric Assay Kit (Cat# CASP3F, Sigma-Aldrich), as previously described63 (link). Further details are described in Supplementary Data.
10
Quantification of Caspase-3 Activity
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Caspase-3 activity was assessed using a Caspase-3 Fluorimetric Assay kit, (Sigma-Aldrich), according to the manufacturer's instructions. Briefly, the cells were seeded into 96-well plates at a density of 10,000 cells/cm2 and incubated with 20 μM 15d-PGJ2 and 1 μM doxorubicin for 24 h. After exposure to the drugs for 24 h, the supernatants were aspirated and the cells were harvested with lysis buffer [50 mM HEPES (pH 7.4), 5 mM CHAPS and 5 mM DTT]. The reaction buffer, including acetyl-Asp-Glu-Val-Asp-7-amido4-methylcoumarin (Ac-DEVD-AMC), a caspase-3 specific substrate, was added to the wells and the production of AMC was sequentially detected with a CytoFluor® Plate reader (MTX Lab Systems, Vienna, VA, USA) at an excitation wavelength of 360 nm and at an emission wavelength of 460 nm. The enzyme activities were determined as initial velocities expressed as nmol AMC/min/ml and were then corrected by the quantity of protein in each well detected by bicinchoninic acid protein assays (Thermo Fisher Scientific, Waltham, MA, USA).
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