Caspase-3, -8 and -9 activities were measured using Caspase-3 (#K106), caspase-8 (#K113), and caspase-9 (#K119) assay kits from BioVision, Inc. (Milpitas, CA, USA). After treatment, cellular proteins were extracted by using cell lysis buffer and then quantified using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc., #23227, Waltham, MA, USA). Subsequently, 150 ∼ 200 ug cellular proteins were reacted with corresponding substrates (DEVD-pNA, IETD-pNA and LEHD-pNA). Caspase-3, -8 and -9 activities were subsequently measured as the optical density of the cleaved substrate at 405 nm using a microplate reader (Bio-Tek Instruments, Inc., #ELX-800, Winooski, VT, USA).
Caspase 9
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Caspase-9 is an enzyme that plays a crucial role in the initiation of the apoptosis (programmed cell death) pathway. It is a member of the caspase family of proteases and is considered a key regulator of the intrinsic (mitochondrial) apoptosis pathway. Caspase-9 functions as an initiator caspase, activating downstream effector caspases that execute the apoptotic process.
Automatically generated - may contain errors
Lab products found in correlation
Caspase 3, by Abcam (66 mentions)
Bcl 2, by Abcam (35 mentions)
Bcl2 associated x (bax), by Abcam (31 mentions)
Cleaved caspase 3, by Abcam (18 mentions)
Cleaved caspase 9, by Abcam (16 mentions)
β actin, by Abcam (15 mentions)
Gapdh, by Abcam (14 mentions)
Caspase 8, by Abcam (10 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (8 mentions)
Pvdf membrane, by Merck Group (8 mentions)
Caspase 12, by Abcam (6 mentions)
Cytochrome c, by Abcam (5 mentions)
P akt, by Abcam (5 mentions)
Cyt c, by Abcam (5 mentions)
Protease inhibitor cocktail, by Merck Group (5 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (5 mentions)
E cadherin, by Abcam (5 mentions)
Mmp 9, by Abcam (5 mentions)
Caspase 3, by Cell Signaling Technology (5 mentions)
P erk, by Cell Signaling Technology (4 mentions)
117 protocols using caspase 9
1
Quantifying Caspase Enzyme Activities
2
Caspase-3 and Caspase-9 Activity Assay
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After the treatment, cells were collected and pellets were suspended in chilled cell lysis buffer. Colorimetric assay kits were used to analyze the activities of caspase-3 and caspase-9 (BioVision, Milpitas, CA, USA). In brief, cytosolic protein from the cells was extracted and protein concentration was determined by BCA assay (Thermo Scientific). In each assay, 50 μL cell lysis buffer containing 50–200 μg protein was used. At the end, absorbance at 405 nm was measured using a microplate reader (BioRad).
3
Mitochondrial Apoptosis Pathway Assay
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AS-IV (>99%) was purchased from the National Institutes for Food and Drug Control, China. The mitochondrial isolation kit, caspase-3, caspase-9, and caspase-8 Colorimetric Assay Kits were obtained from Biovision, USA. The detection kits of GSH and Mn-SOD were obtained from Jiancheng Bioengineering Institute, China. TUNEL assay kit was from KeyGEN Biotech Co. Ltd, China. Protease inhibitors (complete mini tablets) were purchased from Roche, Germany. Antibodies of Bax, Bcl-2, and Bid were from Abcam, Novus, Millipore, USA. The detection kit of MDA and antibodies of AIF and Cyt-c were purchased from Beyotime Institute of Biotechnology, China. The mitochondrial membrane potential fluorescence detection kit was obtained from GENMED, China. Ultrapure RNA extraction kit, Reverse Transcription System, and miRNA Real-Time PCR assay kit were from TaKaRa, Japan.
4
Mechanistic Insights into Anticancer Effects
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Benzo[a]pyrene, curcumin and resveratrol were procured from Sigma Aldrich company. Antibodies p53, phosphorylated[ser-15]-p53 and colorimetric kits for caspase 3 as well as Caspase 9 were procured from Biovision [USA]. 3H labelled thymidine and 14C labeled Glucose were procured from Board of Radiation Isotope Technology [BRIT], Trombay, India. All other reagents were procured from Merck Chemicals and Loba chemicals Pvt. Ltd.
5
Quercetin Modulates Signaling Pathways
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Approximately 2 × 106 cells were managed for 48 h at the concentration of 100 μmol/L quercetin. Western blotting was performed as described previously [4 (link)]. Antibodies against GSK-3β/p-GSK-3β, β-catenin/p-β-catenin, Lef-1, cyclin D1, and BCR-ABL /p-BCR-ABL were provided form Santa Cruz company (CA, USA), PPAR-δ from Millipore (Boston, USA), caspase-3, caspase-8, and caspase-9 from BioVision (San Francisco, USA), β-actin from HaiGene company (Heilongjiang, China).
6
Fluorogenic Caspase Activity Assay
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Direct caspase-4 activation assessment: Caspase-4/11 activity was detected by a fluorogenic caspase substrate Z-VAD-AMC. Z-VAD-AMC was added into the reaction system at a final concentration of 75 mM. The reaction mixture was transferred to a 96-well plate and incubated at 37 C for 30 min. Substrate cleavage was monitored by measuring the fluoresce detected at ex365 nm/em 450 nm on a Synergy H1 Microplate Reader (Bio Tek, Winooski, USA).
For cell lysates samples: caspase activities in bile acids, doxorubicin or LPS transfection treated cells as indicated were determined following the instructions of Caspase Colorimetric Assay Kit (caspase-1, K111; caspase-3, K106; caspase-4, K127; caspase-8, K113; caspase-9, K119, Biovision), and detected by a Synergy H1 Microplate Reader (Bio Tek, Winooski, USA). Caspase activities were indicated as relative folds change to control.
For caspase activity assays in pyroptosome assembling incubation buffer: Caspase substrate (Ac-YVAD-pNA for caspase-1, Ac-LEVD-pNA for caspase-4, Ac-IETD-pNA for caspase-8, Ac-LEHD-pNA for caspase-9, or Ac-DEVD-pNA for caspase-3) was added into reaction buffer to a final concentration of 200 mM, and incubated at 37 C for 2 h. Read samples at 405 nm on a Synergy H1 Microplate Reader (Bio Tek, Winooski, USA). Caspase activities were indicated as relative fold changes to caspase control.
For cell lysates samples: caspase activities in bile acids, doxorubicin or LPS transfection treated cells as indicated were determined following the instructions of Caspase Colorimetric Assay Kit (caspase-1, K111; caspase-3, K106; caspase-4, K127; caspase-8, K113; caspase-9, K119, Biovision), and detected by a Synergy H1 Microplate Reader (Bio Tek, Winooski, USA). Caspase activities were indicated as relative folds change to control.
For caspase activity assays in pyroptosome assembling incubation buffer: Caspase substrate (Ac-YVAD-pNA for caspase-1, Ac-LEVD-pNA for caspase-4, Ac-IETD-pNA for caspase-8, Ac-LEHD-pNA for caspase-9, or Ac-DEVD-pNA for caspase-3) was added into reaction buffer to a final concentration of 200 mM, and incubated at 37 C for 2 h. Read samples at 405 nm on a Synergy H1 Microplate Reader (Bio Tek, Winooski, USA). Caspase activities were indicated as relative fold changes to caspase control.
7
Colorimetric Caspase Assays in Rabbit Liver
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8
Mitochondrial Protein Profiling in Cells
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Cells were lysed in RIPA lysis buffer and phenylmethanesulphonyl fluoride at 4°C for 30 min. Mitochondria were isolated using the Mitochondria Isolation Kit for Cultured Cells (Thermo Scientific, Rockford, IL, USA) and prepared for Western blot analysis. A total of 30 μg of protein was separated in a 6% to 12.5% SDS-PAGE gel, transferred to a PVDF membrane and probed with the following antibodies overnight at 4°C: caspase-9 (cat# ab202068, 1:600 dilution, Epitomics), cytochrome C (cat# ab133504, 1:400 dilution, Epitomics), caspase-12 (cat# 3282-100, 1:1000 dilution, BioVision), VDAC1 (sc-390996, 1:600 dilution, Santa Cruz), Notch1 (cat# 3282-100, 1:400 dilution, Santa Cruz), Notch2 (cat# 5732, 1:1000 dilution, Cell Signaling Technology), Hes1 (cat# ab71559, 1:400 dilution, Abcam) and Hey1 (cat# ab22614, 1:400 dilution, Abcam). After incubation with either HRP-conjugated anti-rabbit or anti-mouse antibodies, immunoreactive bands were visualized by chemiluminescence (ECL Plus; GE Health Care, Piscataway, NJ), and the membranes were exposed to light film (BioMax MR; Kodak, Rochester, NY). The β-actin antibody (1:2000 dilution; Santa Cruz) was used as a loading control.
9
Apoptosis Signaling Pathways Analysis
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MCF-7 cells were placed in a six-well culture plate at a density of 5×105 cells per well and treated with 12.5 µg/mL of each NP or with NPs plus NAC/SP600125 for 24 hours. Then, the cells were lysed using radio immunoprecipitation assay lysis buffer, and the protein concentrations of the cell lysates were measured using a bicinchoninic acid protein assay kit (Beyotime Biotechnology, Jiangsu, People’s Republic of China). Equal amounts of cell lysate protein were resolved in 12% sodium dodecyl sulfate-PAGE gels and transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Primary antibodies against the following proteins were used: JNK (Cell Signaling Technology, Danvers, MA, USA), p-JNK (Cell Signaling Technology), ERK (Cell Signaling Technology), p-ERK (Cell Signaling Technology), p38 (Cell Signaling Technology), p-p38 (Cell Signaling Technology), Bax (Proteintech, Chicago, IL, USA), Bcl-2 (Epitomics, Burlingame, CA, USA), cytochrome-c (Epitomics), caspase-3 (Epitomics), caspase-9 (Epitomics), PARP (Epitomics) and GAPDH (Proteintech).
10
Comprehensive Hepatoprotective Mechanism Study
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DHM (purity ≥99.0%) and TAA (purity ≥99.0%) were purchased from Sigma. Hematoxylin-eosin (H and E), TUNEL and Masson stain kits, and commercial assay kits for ALT and AST, SOD, GSH, MDA were obtained from the Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). The immunofluorescent staining kits and the enhanced chemiluminescence (ECL) kit were purchased from Beyotime science and technology Co., Ltd. (Beijing, China). The antibody of rabbit monoclonal α-SMA, TGF-β1, CYP2E1, Cleaved Caspase-3, Caspase3, Cleaved Caspase-9, Caspase-9, IκB kinase α (IKK-α), IκB kinase β (IKK-β), p-IKKα, p-IKKβ, inhibitor of IκBα (IκBα), p-IκBα, NF-κB, p-NF-κB, PI3K, p-PI3K, Akt, p-Akt, B-associated X (Bax), Bcl-2, β-actin and secondary antibodies for western blot were all obtained from Abcam (Cambridge, United Kingdom).
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