Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit Blue Fluorescence

Detection of Caspase 3/7 activity in Jurkat cells with Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit. Jurkat cells were seeded on the same day at 80,000 cells/well/90 µL in a Costar black wall/clear bottom 96-well plate. The cells were treated with or without 1 µM of staurosporine for 4 hours, and with or without 10 µM of the caspase inhibitor AC-DEVD-CHO for 10 minutes. The caspase 3/7 assay solution (100 µL/well) was added and incubated at room temperature for 1 hour. The fluorescence intensity was measured at Ex/Em = 360/470 nm (Cutoff = 420 nm).

Albumin exposure upregulates pro-apoptotic pathways in podocytes. A, Activated caspase 3/7 activity normalized to total cellular protein in podocytes treated with 5 mg/ml albumin (closed bars) or 5 mg/ml dextran (open bars) as an oncotic control for the indicated amounts of time. * denotes P<0.0001 compared to corresponding dextran treated control. B, TUNEL staining of cultured podocytes treated with 5 mg/ml dextran (Dex) or 5 mg/ml albumin (Alb). Nuclei are stained blue with DAPI. TUNEL-positive nuclei are green. C, Caspase 3/7 activity normalized to total cellular protein in podocytes treated with 5 mg/ml albumin or 5 mg/ml albumin +50 µM z-VAD, a pan-caspase inhibitor. z-VAD largely abrogated caspase activity (*, Pâ€Š=â€Š0.01 versus albumin+z-VAD). D, Percentage of dead cells measured using the trypan blue assay in podocytes treated with 5 mg/ml albumin alone (open bar) or 5 mg/ml albumin +50 µM z-VAD (closed bar) for 24 hrs or 5 mg/ml albumin alone (horizontal hatched bar) or 5 mg/ml albumin +50 µM z-VAD for 48 hrs (vertical hatched bar). * denotes Pâ€Š=â€Š0.001 compared to albumin+z-VAD at 48 hrs. *Caspase activity was determined using the Caspase 3/7 Activity kit (AAT Bioquest, Sunnyvale, CA) according to the manufacturer’s directions. Source: Graph from <strong>Endocytosis of Albumin by Podocytes Elicits an Inflammatory Response and Induces Apoptotic Cell Death</strong> by Kayo Okamura, et al., <em>PLoS ONE</em>, Jan. 2013.

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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Spectral properties

Excitation (nm)	341
Emission (nm)	441

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12352200

Alternative formats

Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Green Fluorescence*

Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Red Fluorescence*

Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Green Fluorescence Optimized for Flow Cytometry*

Overview SDS Protocol

Excitation (nm)

341

Emission (nm)

441

Our Cell Meter™ assay kits are a set of tools for monitoring cell viability. There are a variety of parameters that can be used for monitoring cell viability. This particular kit is designed to monitor cell apoptosis through measuring Caspase 3 activation. Caspase 3 is widely accepted as a reliable indicator for cell apoptosis since the activation of caspase-3 (CPP32/apopain) is important for the initiation of apoptosis. Caspase 3 has substrate selectivity for the peptide sequence Asp-Glu-Val-Asp (DEVD). This kit uses Ac-DEVD-AMC as a fluorogenic indicator for caspase-3 activity. Cleavage of AMC peptides by caspase 3 generates strongly fluorescent AMC that is monitored fluorimetrically at 450-480 nm with excitation of 340-370 nm. The kit provides all the essential components with an optimized assay protocol. The assay is robust, and can be readily adapted for high-throughput assays. Using 100 uL of reagents per well in a 96-well format, this kit provides sufficient reagents to perform 200 assays. Using 25 uL of reagents per well in a 384-well format, this kit provides sufficient reagents to perform 800 assays.

Platform

Fluorescence microplate reader

Excitation	360 nm
Emission	470 nm
Cutoff	420 nm
Recommended plate	Black wall/clear bottom
Instrument specification(s)	Top/Bottom read mode

Components

Example protocol

AT A GLANCE

Protocol summary

Prepare cells with test compounds (100 µL/well/96-well plate or 25 µL/well/384-well plate)
Add equal volume of Caspase 3/7 Substrate working solution
Incubate at room temperature for 1 hour
Monitor fluorescence intensity at Ex/Em = 350/450 nm (Cutoff = 420 nm)

Important notes
Thaw one vial of each kit component at room temperature before starting the experiment.

PREPARATION OF WORKING SOLUTION

Add 50 µL of Caspase 3/7 Substrate (Component A) into 10 mL of Assay Buffer (Component B) and mix well to make Caspase 3/7 Substrate working solution. Note: Aliquot and store the unused Components A and B at -20 ^oC. Avoid repeated freeze/thaw cycles.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

Treat cells by adding 10 µL/well of 10X test compounds (96-well plate) or 5 µL/well of 5X test compounds (384-well plate) into PBS or the desired buffer. For blank wells (medium without the cells), add the same amount of compound buffer.
Incubate the cell plate in a 37°C, 5% CO₂incubator for a desired period of time (4 - 6 hours for Jurkat cells treated with camptothecin) to induce apoptosis.
Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Caspase 3/7 Substrate working solution.
Incubate the plate at room temperature for at least 1 hour, protected from light. Note: If desired, add 1 µL of the 1 mM Ac-DEVD-CHO caspase 3/7 inhibitor into selected samples 10 minutes before adding Caspase 3/7 working solution at room temperature to confirm the inhibition of the caspase 3/7-like activities.
Centrifuge cell plate (especially for the non-adherent cells) at 800 rpm for 2 minutes (brake off).
Monitor the fluorescence intensity with a fluorescence microplate reader at Ex/Em = 350/450 nm (Cutoff = 420 nm).

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Excitation (nm)	341
Emission (nm)	441

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)
Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit Green Fluorescence	500	522	80000
Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit Red Fluorescence	532	619	-

Images

Figure 1. Detection of Caspase 3/7 activity in Jurkat cells with Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit. Jurkat cells were seeded on the same day at 80,000 cells/well/90 µL in a Costar black wall/clear bottom 96-well plate. The cells were treated with or without 1 µM of staurosporine for 4 hours, and with or without 10 µM of the caspase inhibitor AC-DEVD-CHO for 10 minutes. The caspase 3/7 assay solution (100 µL/well) was added and incubated at room temperature for 1 hour. The fluorescence intensity was measured at Ex/Em = 360/470 nm (Cutoff = 420 nm).

Figure 2. Albumin exposure upregulates pro-apoptotic pathways in podocytes. A, Activated caspase 3/7 activity normalized to total cellular protein in podocytes treated with 5 mg/ml albumin (closed bars) or 5 mg/ml dextran (open bars) as an oncotic control for the indicated amounts of time. * denotes P<0.0001 compared to corresponding dextran treated control. B, TUNEL staining of cultured podocytes treated with 5 mg/ml dextran (Dex) or 5 mg/ml albumin (Alb). Nuclei are stained blue with DAPI. TUNEL-positive nuclei are green. C, Caspase 3/7 activity normalized to total cellular protein in podocytes treated with 5 mg/ml albumin or 5 mg/ml albumin +50 µM z-VAD, a pan-caspase inhibitor. z-VAD largely abrogated caspase activity (*, Pâ€Š=â€Š0.01 versus albumin+z-VAD). D, Percentage of dead cells measured using the trypan blue assay in podocytes treated with 5 mg/ml albumin alone (open bar) or 5 mg/ml albumin +50 µM z-VAD (closed bar) for 24 hrs or 5 mg/ml albumin alone (horizontal hatched bar) or 5 mg/ml albumin +50 µM z-VAD for 48 hrs (vertical hatched bar). * denotes Pâ€Š=â€Š0.001 compared to albumin+z-VAD at 48 hrs. *Caspase activity was determined using the Caspase 3/7 Activity kit (AAT Bioquest, Sunnyvale, CA) according to the manufacturer’s directions. Source: Graph from Endocytosis of Albumin by Podocytes Elicits an Inflammatory Response and Induces Apoptotic Cell Death by Kayo Okamura, et al., PLoS ONE, Jan. 2013.

Citations

View all 33 citations: Citation Explorer

Trans-[bis (benzimidazol-2-ylidene) dichlorido] platinum (II) complexes with peculiar modes of action and activity against cisplatin-resistant cancer cells
Authors: B{\"a}r, Sofia I and Schleser, Sebastian W and Oberhuber, Natalie and Herrmann, Alexander and Schlotte, Luca and Weber, Stefanie E and Schobert, Rainer
Journal: Journal of Inorganic Biochemistry (2023): 112028

Involvement of necroptosis in the selective toxicity of the natural compound ($\pm$) gossypol on squamous skin cancer cells in vitro
Authors: Haasler, Lisa and von Montfort, Claudia and Kondadi, Arun Kumar and Golombek, Mathias and Ebbert, Lara and Wenzel, Chantal-Kristin and Stahl, Wilhelm and Reichert, Andreas S and Brenneisen, Peter
Journal: Archives of Toxicology (2023): 1--18

Oxime derivative TFOBO promotes cell death by modulating reactive oxygen species and regulating NADPH oxidase activity in myeloid leukemia
Authors: Jo, Ahyoung and Kwak, Jae-Hwan and Woo, Soo-Yeon and Kim, Bo-Young and Son, Yonghae and Choi, Hee-Seon and Kim, Jayoung and Kwon, Munju and Cho, Hyok-Rae and Eo, Seong-Kug and others,
Journal: Scientific reports (2022): 1--10

Revisiting the anticancer properties of phosphane (9-ribosylpurine-6-thiolato) gold (I) complexes and their 9H-purine precursors
Authors: Kober, Luisa and Schleser, Sebastian W and B{\"a}r, Sofia I and Schobert, Rainer
Journal: JBIC Journal of Biological Inorganic Chemistry (2022): 731--745

Visfatin inhibits colon cancer cell apoptosis and decreases chemosensitivity to 5-FU by promoting the SDF-1/CXCR4/Akt axis
Authors: Zhao, Quan and Long, Yaxin and Cheng, Wen and Huang, Yingguang and Li, Jinyuan and Li, Yuejin and Li, Xing and Guo, Xiaodong and Li, Yu and Li, Guosan and others,
Journal: International journal of oncology (2022): 1--13

Melatonin alleviates alcoholic liver disease via EGFR--BRG1--TERT axis regulation
Authors: Che, Zhaodi and Song, Yali and Xu, Chengfang and Li, Wei and Dong, Zhiyong and Wang, Cunchuan and Ren, Yixing and So, Kwok-Fai and Tipoe, George L and Wang, Fei and others,
Journal: Acta Pharmaceutica Sinica B (2022)

Combination of Goniothalamin and Sol-Gel-Derived Bioactive Glass 45S5 Enhances Growth Inhibitory Activity via Apoptosis Induction and Cell Cycle Arrest in Breast Cancer Cells MCF-7
Authors: Bakar, Siti Aishah Abu and Ali, Abdul Manaf and Noor, Siti Noor Fazliah Mohd and Hamid, Shahrul Bariyah Sahul and Azhar, Nur Asna and Mohamad, Noor Muzamil and Ahmad, Nor Hazwani
Journal: BioMed Research International (2022)

Extracellular vesicle-mediated endothelial apoptosis and EV-associated proteins correlate with COVID-19 disease severity
Authors: Krishnamachary, Balaji and Cook, Christine and Kumar, Ashok and Spikes, Leslie and Chalise, Prabhakar and Dhillon, Navneet K
Journal: Journal of extracellular vesicles (2021): e12117

Knockdown of CENPK inhibits cell growth and facilitates apoptosis via PTEN-PI3K-AKT signalling pathway in gastric cancer
Authors: Wu, Shusheng and Cao, Lulu and Ke, Lihong and Yan, Ying and Luo, Huiqin and Hu, Xiaoxiu and Niu, Jiayu and Li, Huimin and Xu, Huijun and Chen, Wenju and others,
Journal: Journal of Cellular and Molecular Medicine (2021): 8890--8903

microRNA-144 inhibits cell proliferation and invasion by directly targeting TIGAR in esophageal carcinoma
Authors: Mu, Yushu and Wang, Qifei and Tan, Lei and Lin, Lin and Zhang, Benhua
Journal: Oncology letters (2020): 3079--3088