Caspase 3 activity was determined using a caspase 3 activity assay kit, according to the manufacturer's instructions (Biovision, Inc., Milpitas, CA, USA). Briefly, the cells were lysed in caspase 3 sample lysis buffer (Biovision, Inc.). The homogenates were then centrifuged at 10,000 × g and 4°C for 10 min and the supernatant was collected for protein estimation using bicinchoninic acid for the caspase 3 assay. The cell lysates were then exposed to the DEVD substrate conjugate provided in the kit for 1 h at 37°C. The sample was measured in an automatic microplate reader (SpectraMax M5; Molecular Devices, LLC, Sunnyvale, CA, USA) at an excitation of 400 nm and emission of 505 nm.
Caspase 3 activity assay kit
Manufactured by Abcam
Sourced in United States, United Kingdom, China
The Caspase-3 Activity Assay Kit is a laboratory tool designed to measure the activity of the enzyme Caspase-3. Caspase-3 is a key executioner in the apoptosis (programmed cell death) pathway. The kit provides a colorimetric or fluorometric method to quantify Caspase-3 activity in cell and tissue samples.
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Lab products found in correlation
Microplate reader, by Thermo Fisher Scientific (4 mentions)
Caspase 3 sample lysis buffer, by Abcam (2 mentions)
Spectramax m5, by Molecular Devices (2 mentions)
Bradford assay, by Merck Group (2 mentions)
Mitoprobe jc 1, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Dtx 880 multimode detector, by Beckman Coulter (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Abcam (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem with glucose, by Thermo Fisher Scientific (1 mentions)
P gsk3β, by Cell Signaling Technology (1 mentions)
2 7 dichlorodihydrofluorescein diacetate dcfda fluorescence kit, by Merck Group (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
Metformin, by Merck Group (1 mentions)
Ls55 spectrofluorometer, by PerkinElmer (1 mentions)
Sod and mda kit, by Nanjing Jiancheng (1 mentions)
Celltiter glo assay, by Promega (1 mentions)
Apoptag plus in situ apoptosis fluorescein detection kit, by Merck Group (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
44 protocols using caspase 3 activity assay kit
1
Quantifying Caspase 3 Activity
2
Caspase-3 Activity Assay Protocol
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Caspase-3 activity was determined using a caspase-3 activity assay kit, according to the manufacturer's protocol (Biovision, Inc., Milpitas, CA, USA). Briefly, the cells were lysed in caspase-3 sample lysis buffer (Biovision, Inc.). The homogenates were then centrifuged at 10,000 × g at 4°C for 10 min and the supernatant was collected for protein estimation using bicinchoninic acid for the caspase-3 assay. The cell lysates were then exposed to the DEVD substrate conjugate provided in the kit for 1 h at 37°C. The sample was measured using an automatic microplate reader (SpectraMax M5; Molecular Devices, LLC, Sunnyvale, CA, USA) at an excitation of 400 nm and emission of 505 nm.
3
Caspase-3 Activity Assay in Penile Tissue
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Caspase-3 activity in the penile tissue was determined by a Caspase-3 Activity Assay Kit (Biovision K106; Biovision, Milpitas, CA, USA) according to the manufacturer's instructions. Tissues were ground and then incubated with cold lysis buffer on ice for 15 min. The lysed tissues were centrifuged for 10 min at 16 000 g at 4°C; then, the supernatants were collected, and the protein concentrations were calculated. The supernatants were transferred to a 96-well plate containing detection buffer, and Ac-DEVD-pNA was added. After incubation at 37°C for 2 h, absorbance was measured at 405 nm with a microplate reader (Thermo Fisher Scientific). The caspase-3 activity of each sample was calculated according to the standard curve and normalized to the protein concentration.
4
Caspase 3 Activity Quantification
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Caspase 3 activity in SW480, SW480-KO55, SW480-KO16, HCT116 and HCT116-OE cell lines were estimated using a caspase 3 activity assay kit, according to the manufacturer's protocol (BioVision, Inc.). The cells were briefly lysed in a buffer solution containing a caspase 3 sample (BioVision, Inc.). The homogenates of cultured cells were then clarified by differential centrifugation at 10,000 × g, and 4 °C for 10 min and the supernatant was collected. The cell lysates (200 µg) were then introduced to the DEVD substrate conjugate for 2 h at 37 °C followed by supernatant collection. The samples were measured in a microplate reader at an excitation of 405 nm.
5
Caspase-3 Activity Assay in Metformin-Treated Cells
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A Caspase-3 Activity Assay kit (BioVision, Mountain View, CA, USA) was used to measure caspase-3 activity according to the manufacturer's instructions. Cells were plated on 60 mm dishes at a density of 2 × 106 cells/mL and treated with various concentrations of metformin for 24 or 48 h. Cells were washed with PBS and harvested with lysis buffer, which was included in the kit. Cells were incubated on ice for 10 min. Cell lysates were then centrifuged at 4°C and 12,000 × g, and the supernatant was then transferred to a new tube and stored on ice. Protein content was analyzed using the Bradford assay (Sigma, St Louis, MO, USA). Assays were performed in 96-well plates containing 90 μg of protein in 50 μL lysis buffer, followed by the addition of 5 µL 4 mM N-Acetyl-Asp-Glu-Val-Asp p-nitroanilide (DEVD-pNA). The samples were incubated at 37°C for 2 h. Absorbance was measured at 405 nm using a DTX 880 Multimode Detector.
6
Caspase-3 Activity Measurement Protocol
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A caspase-3 activity assay kit (BioVision, Mountain View, CA, USA) was used to measure caspase-3 activity according to the manufacturer’s instructions. Cells were plated on 60 mm dishes at a density of 2×106 cells/mL and treated with various concentrations of simvastatin for 24 or 48 hours. Cells were washed with PBS and harvested with lysis buffer included in the kit. Cells were maintained on ice for 10 minutes. Cell lysates were centrifuged at 4°C and 12,000 g, and the supernatant was transferred to a new tube and stored on ice. Protein content was analyzed using the Bradford assay (Sigma Aldrich). Assays were performed in 96-well plates containing 90 μg of protein in 50 μL lysis buffer, followed by the addition of 4 mM DEVD-pNA (5 μL). The samples were incubated at 37°C for 2 hours. Absorbance was measured at 405 nm using a DTX 880 Multimode Detector (Beckman Coulter).
7
Caspase-3 Activity Assay Protocol
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A caspase-3 activity assay kit (BioVision, China) was used to measure caspase-3 activity. First, the collected cells were processed in lysis buffer containing a protease inhibitor cocktail (Merck, Darmstadt, Germany) for 30 min. After centrifugation, the supernatant containing the total protein samples was prepared for cell lysates. Protein samples (5 µL) were treated with Ac-DEVD-AMC agent and read using a Polarstar spectrofluorimeter at 460 nm [21 (link)].
8
Quantifying Caspase-3 Activity in Cells
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Caspase-3 activity was detected by using caspase-3 activity assay kit (Biovision). This assay is a Fluorometric assay which measures caspase-3 activities. The level of caspase-3 activity is measured by detection of cleavage of substrate DEVD-AFC. Briefly, Primary culture cells (1.5 × 105 cells/well) or ovarian cancer cells (2.5 × 105 cells/well) were seeded in 12 well plates or 6 well plates and incubated for 24 hours at 37°C. The cells were treated with 10058-F4 at different concentration for 24 hours and then lysated with lysis buffer. 50 ug cell lysates was used for measuring caspase-3 activity following manufacture instructions. The samples were reading in 96 well plates at a fluorometric plate reader.
9
SKOV3 and Hey Cell Apoptosis Assay
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10058-F4 (F4, Sigma, St. Louis, MO, USA) was dissolved in dimethylsulfoxide (DMSO) according the manufacturer’s instructions and further diluted to indicated concentrations in culture medium before use. SKOV3 cells were maintained in DMEM RPMI-1640 culture medium supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, and 100 mg/mL streptomycin. Hey cells were cultured in RPMI-1640 medium with 5% FBS. Fetal bovine serum (FBS) was from Invitrogen (Carlsbad, CA, USA). The Annexin V-FITC Apoptosis Detection kit and Caspase3 Activity Assay kit were purchased from Biovision (Mountain View, CA, USA). All antibodies were purchased from Cell Signaling (Boston, MA, USA). All other materials were obtained from Life Technologies or from Sigma-Aldrich.
10
Metformin Modulates Cell Signaling
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Dulbecco’s Modified Eagle’s Medium (DMEM) with glucose and without glucose and all medium additives were obtained from Gibco BRL (Grand Island, NE, USA). Methylthiazolyldiphenyl-tetrazolium bromide (MTT), glucose, metformin, N-acetyl-l -cysteine (NAC), FH535, and 2-7-Dichlorodihydrofluorescein diacetate (DCFDA) fluorescence kit were purchased from Sigma Aldrich (St. Louis, MI, USA). PD98059 was purchased from Calbiochem (La Jolla, CA, USA). LDH, Caspase-3 activity assay kit and ATP measurement kits were purchased from BioVision (Milpitas, CA, USA). The antibodies specific for ERK, p-ERK, GSK3β, p-GSK3β, and PARP were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibodies specific for Bax and Bcl-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and the antibody specific for GAPDH was purchased from Thermo Fisher (Rockford, IL, USA).
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