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Active caspase 3 apoptosis kit

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The Active Caspase-3 Apoptosis Kit is a laboratory tool designed to detect and quantify the active form of the Caspase-3 enzyme, which is a key mediator of apoptosis, or programmed cell death. The kit provides the necessary reagents and protocols to measure the activity of active Caspase-3 in cellular samples.

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Lab products found in correlation

Celltrace violet, by Thermo Fisher Scientific (4 mentions) Annexin 5 apoptosis detection kit, by Thermo Fisher Scientific (2 mentions) Monensin, by Thermo Fisher Scientific (2 mentions) Anti cd71 r17217, by Thermo Fisher Scientific (2 mentions) Anti cd98 rl388, by Thermo Fisher Scientific (2 mentions) P s6 d57.2.2e, by Cell Signaling Technology (2 mentions) C myc d84c12, by Cell Signaling Technology (2 mentions) Bim c34c5, by Cell Signaling Technology (2 mentions) Lsrfortessa, by BD (2 mentions) Annexin 5 fitc, by BD (2 mentions) Accuri c6, by BD (1 mentions) Acetylcholinesterase, by Merck Group (1 mentions) Annexin 5 fitc assay kit, by BD (1 mentions) Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions) Phorbol 12 myristate 13 acetate, by Merck Group (1 mentions) Il 1β, by MyBioSource (1 mentions) Interleukin 6 (il 6), by MyBioSource (1 mentions)

Close spelling variants of this product

PE-conjugated active caspase-3 apoptosis kit FITC Active Caspase-3 Apoptosis Kit PE-conjugated monoclonal active-caspase-3 antibody apoptosis kit PE Active Caspase-3 Apoptosis Kit Caspase-3 (active form) apoptosis kit FITC-conjugated Monoclonal Active Caspase-3 Antibody Apoptosis Kit I Active caspase-3 Caspase-3 Apoptosis kits

14 protocols using active caspase 3 apoptosis kit

1

Evaluating Cell Cycle and Apoptosis

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Flow cytometric analysis (BD Accuri™ C6, BD biosciences) was performed to evaluate cell cycle distribution by propidium iodide (PI) staining, apoptosis by AnnexinV-FITC/PI staining, and to detect acidic vesicular organelles (AVOs) by acridine orange staining, as previously described
[39 (link), 40 (link)]. Active Caspase-3 Apoptosis Kit (BD biosciences) was used to detect the heterodimer of 17 and 12 kDa subunits, which is derived from the pro-enzyme.
Del Bufalo D., Desideri M., De Luca T., Di Martile M., Gabellini C., Monica V., Busso S., Eramo A., De Maria R., Milella M, & Trisciuoglio D. (2014). Histone deacetylase inhibition synergistically enhances pemetrexed cytotoxicity through induction of apoptosis and autophagy in non-small cell lung cancer. Molecular Cancer, 13, 230.
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2

Apoptosis and Proliferation Assays

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The Annexin V Apoptosis Detection Kit (eBioscience) and the Active Caspase 3 Apoptosis Kit (BD Pharmingen) were used for apoptosis assays. WEHI-control and WEHI-miR148a cells were stimulated with 2 µg/ml anti-IgM for the indicated amounts of time or left unstimulated, harvested, stained with both apoptosis kits following the manufacturer’s protocols, and analyzed by flow cytometry for the markers annexin V and active caspase 3. For proliferation analysis, cells were labeled with the fluorescent dye Cell Trace Violet (Life Technologies) following the manufacturer’s instructions, stimulated with 2 µg/ml anti-IgM for the indicated amounts of time or left unstimulated, and the dilution of the dye determined by flow cytometry.
Gonzalez-Martin A., Adams B.D., Lai M., Shepherd J., Salvador-Bernaldez M., Salvador J.M., Lu J., Nemazee D, & Xiao C. (2016). The microRNA miR-148a functions as a critical regulator of B cell tolerance and autoimmunity. Nature immunology, 17(4), 433-440.
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3

Comprehensive Immune Cell Profiling

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For analysis of surface markers, cells were stained in PBS containing 2% (wt/vol) BSA, with anti-CD4 (RM4-5), anti-CD8α (53-6.7), anti-TCRβ (H57-597), anti-CD44 (1M7), anti-CD62L (MEL-14), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD71 (R17217), and anti-CD98 (RL388; all from eBioscience). Intracellular Foxp3 (FJK-16s), Ki67 (SolA15), IFN-γ (XMG1.2), IL-4 (11B11), IL-17 (17B7; all from eBioscience), Bim (C34C5), c-Myc (D84C12), and p-S6 (D57.2.2E; all from Cell Signaling Technology) were analyzed by flow cytometry according to the manufacturer’s instructions. For intracellular cytokine staining, T cells were stimulated for 4 h with PMA plus ionomycin in the presence of monensin before intracellular staining according to the manufacturer’s instructions (eBioscience). Caspase-3 activity was measured using active caspase-3 apoptosis kit (BD Biosciences). To monitor cell division, lymphocytes were labeled with CellTrace™ violet (Life Technologies). Flow cytometry data were acquired on LSRII or LSR Fortessa (BD Biosciences) and analyzed using Flowjo software (Tree Star).
Wei J., Long L., Yang K., Guy C., Shrestha S., Chen Z., Wu C., Vogel P., Neale G., Green D.R, & Chi H. (2016). Autophagy enforces functional integrity of regulatory T cells by coupling environmental cues and metabolic homeostasis. Nature immunology, 17(3), 277-285.
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4

Apoptosis and Proliferation Assays

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The Annexin V Apoptosis Detection Kit (eBioscience) and the Active Caspase 3 Apoptosis Kit (BD Pharmingen) were used for apoptosis assays. WEHI-control and WEHI-miR148a cells were stimulated with 2 µg/ml anti-IgM for the indicated amounts of time or left unstimulated, harvested, stained with both apoptosis kits following the manufacturer’s protocols, and analyzed by flow cytometry for the markers annexin V and active caspase 3. For proliferation analysis, cells were labeled with the fluorescent dye Cell Trace Violet (Life Technologies) following the manufacturer’s instructions, stimulated with 2 µg/ml anti-IgM for the indicated amounts of time or left unstimulated, and the dilution of the dye determined by flow cytometry.
Gonzalez-Martin A., Adams B.D., Lai M., Shepherd J., Salvador-Bernaldez M., Salvador J.M., Lu J., Nemazee D, & Xiao C. (2016). The microRNA miR-148a functions as a critical regulator of B cell tolerance and autoimmunity. Nature immunology, 17(4), 433-440.
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5

Comprehensive Immune Cell Profiling

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For analysis of surface markers, cells were stained in PBS containing 2% (wt/vol) BSA, with anti-CD4 (RM4-5), anti-CD8α (53-6.7), anti-TCRβ (H57-597), anti-CD44 (1M7), anti-CD62L (MEL-14), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD71 (R17217), and anti-CD98 (RL388; all from eBioscience). Intracellular Foxp3 (FJK-16s), Ki67 (SolA15), IFN-γ (XMG1.2), IL-4 (11B11), IL-17 (17B7; all from eBioscience), Bim (C34C5), c-Myc (D84C12), and p-S6 (D57.2.2E; all from Cell Signaling Technology) were analyzed by flow cytometry according to the manufacturer’s instructions. For intracellular cytokine staining, T cells were stimulated for 4 h with PMA plus ionomycin in the presence of monensin before intracellular staining according to the manufacturer’s instructions (eBioscience). Caspase-3 activity was measured using active caspase-3 apoptosis kit (BD Biosciences). To monitor cell division, lymphocytes were labeled with CellTrace™ violet (Life Technologies). Flow cytometry data were acquired on LSRII or LSR Fortessa (BD Biosciences) and analyzed using Flowjo software (Tree Star).
Wei J., Long L., Yang K., Guy C., Shrestha S., Chen Z., Wu C., Vogel P., Neale G., Green D.R, & Chi H. (2016). Autophagy enforces functional integrity of regulatory T cells by coupling environmental cues and metabolic homeostasis. Nature immunology, 17(3), 277-285.
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6

Apoptosis and Inflammation Assays

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Acetylcholinesterase and phorbol 12-myristate 13-acetate were purchased from Sigma (St. Louis, MO, United States); FITC Annexin V assay kit, active Caspase-3 apoptosis kit and JC-1 were purchased from BD Biosciences (San Jose, CA, United States); IL-1β, IL-6, and TNF-α were purchased from MyBioSource (San Diego, CA, United States); IL-8 and P-selectin ELISA kits were purchased from ThermoFisher (Waltham, MA, United States); IMDM, RPMI1640, FBS, and BSA were purchased from Gibco (Waltham, MA, United States); tanshinone IIA was purchased from Shanghai Pharmaceuticals (Shanghai, China); TPO was purchased from PeproTech (Rocky Hill, NJ, United States).
Chen H., Shu H., Su W., Li B., Zhang H., Li L., Lin C., Yi W., Zhan X.Y., Chen C., Li X., Yang Y., Zhou M, & Yang M. (2022). Tanshinone IIA Has a Potential Therapeutic Effect on Kawasaki Disease and Suppresses Megakaryocytes in Rabbits With Immune Vasculitis. Frontiers in Cardiovascular Medicine, 9, 873851.
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7

Evaluating PARP Inhibitor and Cytokine-Mediated Tumor Cell Lethality

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For in vitro analysis of PARP inhibitor and cytokine-mediated tumor cell lethality, 1 104 murine tumor cells per well were plated in 24-well plates and exposed to increasing concentrations of the PARP inhibitor alone or with IFNγ or TNFα with a final volume of 1 mL/well. Human ovarian cancer cell lines UWB1.289wt and UWB1.289þBRCA1 were plated at 1×105 cells per well due to their slower growth rate. After 72 hours, samples were retrieved using a 1:1 mix of cellstripper (Corning) and trypsin–EDTA (Corning) to release adherent cells, and centrifuged for 3 minutes at 1,500 rpm. Cells were then washed in PBS, stained with the fixable viability dye ZombieNIR (BioLegend) for 10 minutes, and fixed for analysis. For cells stained intracellularly for active caspase-3, the manufacturer's instructions were followed (active Caspase-3 apoptosis kit; BD Biosciences).
Higuchi T., Flies D.B., Marjon N.A., Mantia-Smaldone G., Ronner L., Gimotty P.A, & Adams S.F. (2015). CTLA-4 Blockade Synergizes Therapeutically with PARP Inhibition in BRCA1-Deficient Ovarian Cancer. Cancer immunology research, 3(11), 1257-1268.
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8

Apoptosis Analysis Using Annexin-V and Caspase-3

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Apoptosis analysis was performed by staining the cells with Annexin-V and PI (Thermo Fisher Scientific) according to the manufacturer’s instructions and analyzed using flow cytometry. Briefly, the cells were collected and washed with ice-cold PBS. The cells were stained with anti -annexin V antibody and propidium iodide (PI) and incubated in dark for 15 mins at room temperature. The stained cells were analyzed with FACS Calibur.
Cell death was further confirmed by staining for active form of caspase-3 and analysis through flow cytometry. Active caspase-3 staining was performed using Active caspase-3 apoptosis kit (BD biosciences) according to the manufacturer’s instructions. After fixation and permeabilization with BD cytofix/cytoperm fixation/permeabilization buffer, the cells were stained with fluorescent conjugated anti-active caspase 3 antibody. The cells were incubated in dark at room temperature for 30 mins, washed and analyzed with FACS Calibur.
Kumar A., Bhattacharyya J, & Jaganathan B.G. (2017). Adhesion to stromal cells mediates imatinib resistance in chronic myeloid leukemia through ERK and BMP signaling pathways. Scientific Reports, 7, 9535.
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9

Culturing Bovine Meat Buds in Collagen

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Cultured bovine Ha2/5+ cells were seeded and cultured in EZ sphere dishes of diameter ~500 µm and depth ~200 µm (#4020-900SP, IWAKI, Tokyo, Japan). A meat bud was used in the experiment after culturing for three days. For embedding meat buds in collagen gel, 1% BSA/PBS (5 mL) was placed into a six-well plate and incubated for blocking (30 min). The pH of collagen (Collagen I, Rat Tail, A10483, Gibco, NC, USA) was adjusted to pH 7.0 on ice. CD29-positive meat buds and collagen were mixed on ice at a ratio of 1:1 and seeded in a six-well plate. To investigate cell apoptosis, meat buds were retrieved from collagen gel with collagenase and analyzed by flow cytometer using Active Caspase-3 Apoptosis Kit (#550480, BD Pharmingen, San Diego, CA, USA).
Naraoka Y., Mabuchi Y., Yoneyama Y., Suto E.G., Hisamatsu D., Ikeda M., Ito R., Nakamura T., Takebe T, & Akazawa C. (2021). Isolation and Characterization of Tissue Resident CD29-Positive Progenitor Cells in Livestock to Generate a Three-Dimensional Meat Bud. Cells, 10(9), 2499.
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10

Apoptosis Analysis by Flow Cytometry

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Cell cycle analysis was performed on 5 × 105 fixed cells resuspended in a RNase A (3 mg/mL) and propidium iodide (PI) solution (1 mg/mL). Samples were analyzed with a C6 Accuri BD (Becton Dickinson & Co., San Jose, CA, USA). The percentage of apoptosis was measured analyzing sub-G1 peaks. Apoptotic cell death was also evaluated by using annexin V–fluorescein isothiocyanate (FITC) apoptosis kit (BD Bioscience) and assayed according to the manufacturer’s instructions. Cells double-stained for both annexin V and PI were analyzed by flow cytometry. PI was used in conjunction with annexin V–FITC to distinguish cells in the earlier stages of apoptosis (annexin V-FITC positive, PI negative) from those in later stages of apoptosis or that were already dead (annexin V–FITC positive, PI positive). Percentage of cells positive for caspase-3 activation was evaluated by Active Caspase-3 Apoptosis Kit (BD biosciences, Milan, Italy) according to the manufacturer’s instructions.
Zwergel C., Fioravanti R., Stazi G., Sarno F., Battistelli C., Romanelli A., Nebbioso A., Mendes E., Paulo A., Strippoli R., Tripodi M., Pechalrieu D., Arimondo P.B., De Luca T., Del Bufalo D., Trisciuoglio D., Altucci L., Valente S, & Mai A. (2020). Novel Quinoline Compounds Active in Cancer Cells through Coupled DNA Methyltransferase Inhibition and Degradation. Cancers, 12(2), 447.
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