To assess the ability of resveratrol to induce the apoptotic process, an ICC reaction was performed. The cells of the tested lines were plated on Millicell EZ SLIDES eight-well glass slides (Merck Millipore, Gernsheim, Germany) in the following amounts: EPP85-181P—1 × 104 cells/well, EPP85-181RNOV—1 × 104 cells/well, AsPC-1—1.5 × 104 cells/well and H6c7—1.5 × 104 cells/well. 24 h later, cells were treated with resveratrol at concentrations of 0, 25, 50 and 100 µM for 48 h. After this time, cells were fixed with methanol-acetone (1:1) for 10 min at 4 °C. The ICC reaction was performed on an Autostainer Link48 (Dako, Glostrup, Denmark). The following primary antibodies were used: Bcl-2 (Dako, Glostrup, Denmark), Bax (Santa Cruz Biotechnology, Dallas, TX, USA) and activated Caspase-3 (Cell Signaling Technology, Boston, MA, USA). Slides were first incubated with primary antibodies against Bcl-2 (ready-to-use), Bax (1:25) and activated Caspase-3 (1:400) for 20 min at room temperature, followed by 20 min with EnVision FLEX/HRP (Dako, Glostrup, Denmark). In the next step, the slides were incubated for 10 min with 3,3’-diaminobenzidine (DAB, Dako. Glostrup, Denmark). The slides were counterstained with EnVision FLEX Hematoxylin (Dako, Glostrup, Denmark) and sealed with coverslips in a mounting medium. The ICC reaction was assessed using a BX-41 light microscope (Olympus, Tokyo, Japan).
Activated caspase 3
Manufactured by Cell Signaling Technology
Sourced in United States
Activated caspase-3 is a laboratory product that detects the presence of the cleaved, active form of caspase-3 protein. Caspase-3 is a crucial effector caspase that plays a central role in the execution phase of cell apoptosis (programmed cell death).
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (3 mentions)
Phospho histone h3, by Cell Signaling Technology (2 mentions)
α tubulin, by Merck Group (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
γh2ax, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Bcl 2, by Agilent Technologies (1 mentions)
Bcl2 associated x (bax), by Agilent Technologies (1 mentions)
Envision flex hrp, by Agilent Technologies (1 mentions)
Millicell ez slides eight well glass slides, by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Bx41 light microscope, by Olympus (1 mentions)
Envision flex hematoxylin, by Agilent Technologies (1 mentions)
Autostainer link 48, by Agilent Technologies (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
Ds fi1 digital camera, by Nikon (1 mentions)
Ultralow adhesion dishes, by Corning (1 mentions)
Eclipse 80i microscope, by Nikon (1 mentions)
Axio observer, by Zeiss (1 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
29 protocols using activated caspase 3
1
Resveratrol-Induced Apoptosis Analysis by ICC
2
Evaluation of Autophagy and Apoptosis in MOSE-L Spheroids
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MOSE-L cells were seeded at a density of 0.5 × 105 cells per well in ultralow-adhesion dishes (Corning). Culture medium was changed every 3 days. After 12 days in culture, the spheroids were harvested, gently centrifuged, rinsed in cold PBS and fixed in 10% buffered formalin, embedded into paraffin, and sectioned at 4–5 µm for immunohistochemical analyses of LC3 (Abgent, San Diego, CA, USA) and activated Caspase-3 (Cell Signaling) as described [75 (link)]. Images were taken using the Nikon Eclipse 80i microscope equipped with a DS-Fi1 Digital Camera and Nikon NIS Elements® 5.42.03 acquisition and quantitation software. All images were processed with Adobe Photoshop Elements® 20.0.
3
Immunofluorescent Analysis of Apoptosis Markers
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Samples grown over Nunc Lab-Tek chambers (Thermo Fisher Scientific, Waltham, MA, USA) were washed with PBS, fixed with 4% paraformaldehyde for 15 min, washed three times with PBS and blocked with PBS 1% BSA-0.3% Triton for 60 min. Samples were then incubated overnight at 4 °C with primary antibody against BAX-6A7 (BD Bioscience, Franklin Lakes, NJ, USA), activated Caspase 3 (Cell Signaling, Danvers, MA, USA) or γH2AX (Cell Signaling, Danvers, MA, USA) in PBS 1% BSA-0.3% Triton.
After three washes with PBS, samples were incubated with the corresponding FITC-conjugated (Activated Caspase-3 or γH2AX) or TRITC-conjugated (BAX-6A7) secondary antibody (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 1 h. Samples were washed three times with PBS and cell nuclei were finally stained with DAPI (2 μg/mL). Images were captured using a ZEISS Axio Observer (ZEISS, Oberkochen, Germany) microscope and analyzed using the Carl Zeiss Microscopy GmbH’s ZEN 3.0 software.
After three washes with PBS, samples were incubated with the corresponding FITC-conjugated (Activated Caspase-3 or γH2AX) or TRITC-conjugated (BAX-6A7) secondary antibody (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 1 h. Samples were washed three times with PBS and cell nuclei were finally stained with DAPI (2 μg/mL). Images were captured using a ZEISS Axio Observer (ZEISS, Oberkochen, Germany) microscope and analyzed using the Carl Zeiss Microscopy GmbH’s ZEN 3.0 software.
4
Endothelial Cell Protein Expression
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Unless otherwise specified, all reagents and chemicals were purchased from Sigma-Aldrich. Antibodies purchased were SIRT1, vascular cell adhesion molecule (VCAM)-1, and activated caspase 3 (Cell Signaling Technology), PEPCK (Cayman Chemical Company), F4/80 (Abcam), DBC1 (Bethyl Laboratories), p53 and p21 (Santa Cruz Biotechnology, Inc.), and actin (Sigma-Aldrich). Human aortic endothelial cells and EGM2 culture media were purchased from Lonza.
5
MTT and PI Assay for Apoptosis
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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). TNF-α, JNK, Bax and activated caspase-3 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-α-tubulin mAb was purchased from Neo Markers (Fremont, CA, USA). The Hybond-P polyvinylidene difluoride membrane, enhanced chemiluminescence (ECL) western blotting detection reagent and analysis system, horseradish peroxidase (HRP)-conjugated donkey anti-rabbit immunoglobulin G (IgG) and sheep anti-mouse IgG were purchased from Amersham (Buckinghamshire, UK).
6
Fractionation and Immunoblotting of Cellular Proteins
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Cell fractions were lysed in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, and 1 mM EDTA supplemented with anti-protease and anti-phosphatase cocktails (Roche). Mitochondrial fraction was obtained with the help of a kit from Pierce. The protein concentration was determined using the Bio-Rad DC kit, and 70 μg of protein was loaded in linear SDS-PAGE gels. After blotting, nitrocellulose filters were probed with primary antibodies against CD47 (clone 2D3; eBioscience), activated caspase-3 (9661; Cell Signaling Technology), PLCγ1 (clone D9H10; Cell Signaling Technology), PLCγ1-Y783 (2821; Cell Signaling Technology), Cox IV (clone 1D6E1A8; Life Technologies), DRP1/DLP1 (clone 8/DLP1; BD Biosciences), and α-tubulin (clone B-5–1–2; Sigma). Immunoreactive proteins were detected using HRP-conjugated secondary antibodies and visualized with the ECL system (Thermo Scientific). Immunoblot images were acquired on a MF-ChemiBIS 4.2 (DNR Bio-Imaging Systems). PLCγ1-Y783 was quantified using Multi Gauge 3.0 software (Fujifilm Life Sciences). The optical density was normalized to the background and was expressed relative to the untreated cells (set at 1.0).
7
Immunohistochemical Analysis of BET Inhibition
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IHC was performed as we described previously (7 (link)). Xenograft tumor tissues were obtained from Balb/c tumor-bearing mice treated with single dose of 5 mg/kg BETd-260 or vehicle control. The following antibodies were used for IHC: BRD2 (IHC-00612), BRD4 (HC-00396), BAD (A302-384A) from Bethyl Laboratories (Shanghai, China); BRD3 (ab264420) from Abcam (Shanghai, China); cleaved PARP (Asp214) (#32563), activated caspase-3 (#9664), and Ki-67 (8D5) (9449) from Cell Signaling Technology (CST, Shanghai, China), Anti-Mcl-1 (MAB828) from R&D Systems (R&D Systems, Shanghai, China).
8
Comprehensive Immunohistochemical Analyses of Intestinal Tissues
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IHC and IF were performed as described previously (Dai et al., 2000 (link)), using formalin-fixed and paraffin-embedded jejunal sections and the following antibodies: human p16 (JC2, Jim Koh, Duke University), mouse p16 (M156, Santa Cruz Biotechnology, Santa Cruz, CA, USA) BrdU (IHC: Becton Dickinson, #347580; IF: Abgene), activated Caspase 3 (#9661, Cell Signaling Technology, Danvers, MA, USA), lysozyme (Dako, # EC.3.2.1.17), chromogranin A (Immunostar, #20085), PCNA (#2586, Cell Signaling), phospho-histone H3 (#9071, Cell Signaling), cyclin D1 (sc-753 (cross-reacts with cyclin D2, Santa Cruz), Cdk4 (#sc-260 (Santa Cruz), and γH2AX (#05-636, EMD Millipore, Billerica, MA, USA). Lgr5-lacZ expression was assayed by a modification of methods described previously (Barker et al., 2007 (link)). Freshly excised tissue was fixed in 2% neutral buffered formalin/0.2% glutaraldehyde/0.01% deoxycholate/0.2% NP40 for 30 min at RT, then incubated in X-gal substrate at RT overnight in the dark before embedding in paraffin. SAβgal staining was at pH 6.0. For p16 IB and Cdk4 IP, intestinal epithelial cells were isolated by treatment of excised tissue with EDTA (Weiser, 1973 (link)) and protein extraction with E1a lysis buffer (Harlow et al., 1985 (link)). For IB of γH2AX, mucosa was scraped off with a razor blade and chromatin extracts sheared by sonication.
9
Cell Lysis and Protein Detection
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Cell lysis was carried out identically for SH-SY5Y cells, HEK293 cells and human fibroblasts as well as MEFs. Cells were washed with phosphate-buffered saline (PBS) and detached with Trypsin at 37°C for 3 min. After 5 min of centrifugation at 14 000 r.p.m., cells were lysed in buffer containing 0.1% Triton X-100 in PBS and 1 × protease inhibitor cocktail (Roche, Penzberg, Germany). Proteins were detected by using antibodies against mortalin (anti-GRP75, Santa Cruz Biotechnology, Dallas, TX, USA), β-Actin (Sigma-Aldrich, Munich, Germany), GAPDH (Life Technologies, Carlsbad, CA, USA), Tom20 (Santa Cruz Biotechnology), Hsp60 (from A Azem, Tel-Aviv University, Ramat Aviv, Israel), activated caspase 3 (Cell Signaling Technology, Danvers, MA, USA), activated caspase 9 (Cell Signaling Technology), Flag (Sigma-Aldrich), α-tubulin (Sigma-Aldrich), β-Tubulin (anti-β-Tubulin, (E7), Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA), GAPDH (anti-GAPDH (G9545), Sigma-Aldrich) and rodent OXPHOS (no. MS604 Mitoscience (Abcam), Cambridgeshire, UK). Secondary antibodies were purchased from GE Healthcare (Buckinghamshire, UK).
Densitometry from western blot was performed using the ImageJ 1.41o software (Wayne Rasband; National Institutes of Health, Bethesda, MD, USA).
Densitometry from western blot was performed using the ImageJ 1.41o software (Wayne Rasband; National Institutes of Health, Bethesda, MD, USA).
10
Regulation of Cell Cycle and Apoptosis
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