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

Detection of Caspase 3/7 Activities in Jurkat cells. Jurkat cells were seeded on the same day at 200,000 cells/90 µL/well in a Costar black wall/clear bottom 96-well plate. The cells were treated with or without 1 µM of staurosporine for 5 hours. The caspase 3/7 working solution (100 µL/well) was added and incubated at room temperature for 1 hour. The fluorescence intensity was measured at Ex/Em = 540/620 nm (Cutoff = 610 nm) with FlexStation fluorescence microplate reader (Molecular Devices).
Detection of Caspase 3/7 Activities in Jurkat cells. Jurkat cells were seeded on the same day at 200,000 cells/90 µL/well in a Costar black wall/clear bottom 96-well plate. The cells were treated with or without 1 µM of staurosporine for 5 hours. The caspase 3/7 working solution (100 µL/well) was added and incubated at room temperature for 1 hour. The fluorescence intensity was measured at Ex/Em = 540/620 nm (Cutoff = 610 nm) with FlexStation fluorescence microplate reader (Molecular Devices).
Detection of Caspase 3/7 Activities in Jurkat cells. Jurkat cells were seeded on the same day at 200,000 cells/90 µL/well in a Costar black wall/clear bottom 96-well plate. The cells were treated with or without 1 µM of staurosporine for 5 hours. The caspase 3/7 working solution (100 µL/well) was added and incubated at room temperature for 1 hour. The fluorescence intensity was measured at Ex/Em = 540/620 nm (Cutoff = 610 nm) with FlexStation fluorescence microplate reader (Molecular Devices).
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Spectral properties
Excitation (nm)532
Emission (nm)619
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


See also: Caspases
Excitation (nm)
532
Emission (nm)
619
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 Z-DEVD-ProRed™ as the fluorogenic indicator for caspase-3 activity. Cleavage of ProRed™ DEVD blocking peptide residue by caspase 3 generates strongly red fluorescent ProRed™ that is monitored fluorimetrically at ~620 nm with excitation of ~530 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 100 assays. Using 25 uL of reagents per well in a 384-well format, this kit provides sufficient reagents to perform 400 assays.

Platform


Fluorescence microplate reader

Excitation540 nm
Emission620 nm
Cutoff610 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Top or bottom read mode

Components


Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells with test compounds (100 µL/well/96-well plate or 25 µL/well/384-well plate)
  2. Add equal volume of Caspase 3/7 Substrate working solution (100 µL/well/96-well plate or 25 µL/well/384-well plate)
  3. Incubate at room temperature for 1 hour
  4. Monitor fluorescence intensity (top or bottom read mode) at Ex/Em = 540/620 nm (Cutoff = 610 nm)

Important notes
Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Z-DEVD-ProRed™ stock solution (200X):
Add 65 µL of DMSO into the vial of Z-DEVD-ProRed™ (Component A) to make 200X Z-DEVD-ProRed™ stock solution. Protect from light.

PREPARATION OF WORKING SOLUTION

Add 50 μL of 200X Z-DEVD-ProRed™ stock solution 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 Z-DEVD-ProRed™ Stock solution (from Step 2.2) and Assay Buffer (Component B) at -20 oC. Avoid repeated freeze/thaw cycles.

SAMPLE EXPERIMENTAL PROTOCOL

Prepare cells:

  • For adherent cells: Plate cells overnight in growth medium at 20,000 cells/well/90 uL for a 96-well or 5,000cells/well/ 20 uL for a 384-well plate black wall/clear bottom plate.

  • For non-adherent cells: Centrifuge the cells from the culture medium and then suspend the cell pellet in culture medium at 80,000 to 200,000 cells/well/90 uL for a 96-well or 20,000 to 50,000 cells/well/20 uL for a 384-well black wall/clear bottom plate. Centrifuge the plate at 800 rpm for 2 minutes with brake off prior to the experiments. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for apoptosis induction

Sample Protocol:

  1. 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.

  2. Incubate the cell plate in a 37°C, 5% CO2, incubator for a desired period of time (3 - 4 hours for Jurkat cells treated with staurosporine) to induce apoptosis.

  3. Add 100 µL/well/96-well or 25 µL/well/384-well plate of Caspase 3/7 Substrate working solution.

  4. 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 to 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.

  5. Monitor the fluorescence intensity with a fluorescence microplate reader (top or bottom read mode) at Ex/Em = 540/620 nm (Cutoff = 610 nm). Note: Sometimes, bottom read gives better signal to background ratio. Centrifuge cell plate (especially for the non-adherent cells) at 800 rpm for 2 minutes (brake off) if using bottom read mode.

Spectrum


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spectrum

Spectral properties

Excitation (nm)532
Emission (nm)619

Images


Citations


View all 28 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
Hexamethylene Amiloride synergizes with Venetoclax to induce lysosome-dependent cell death in acute myeloid leukemia
Authors: Jiang, Xinya and Huang, Kexiu and Sun, Xiaofan and Li, Yue and Hua, Lei and Liu, Fangshu and Huang, Rui and Du, Juan and Zeng, Hui
Journal: iScience (2023)
GONIOTHALAMIN INHIBITS CELL GROWTH, PERTURBS CELL CYCLE AND INDUCES APOPTOSIS IN HUMAN OSTEOSARCOMA SAOS-2 CELLS: Received 2023-01-03; Accepted 2023-02-15; Published 2023-06-06
Authors: Bakar, Siti Aishah Abu and Azhar, Nur Asna and Mohamad, Noor Muzamil and Nordin, Ira Maya Sophia and Ali, Abdul Manaf and Noor, Siti Noor Fazliah Mohd and Hamid, Shahrul Bariyah Sahul and Ahmad, Nor Hazwani
Journal: Journal of Health and Translational Medicine (JUMMEC) (2023): 105--115
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
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)
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
The potential role of extracellular vesicles in COVID-19 associated endothelial injury and pro-inflammation
Authors: Krishnamachary, Balaji and Cook, Christine and Spikes, Leslie and Chalise, Prabhakar and Dhillon, Navneet K
Journal: medRxiv (2020)