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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).
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Catalog Number22797
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
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


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


Component A: Z-DEVD-ProRed™1 vial
Component B: Assay Buffer1 bottle (10 mL)

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


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)532
Emission (nm)619

Citations


View all 22 citations: Citation Explorer
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)
Homeostatic effects of exosomal KLF2 microRNAs: implications for treatment of pulmonary hypertension
Authors: Sindi, Hebah Adel
Journal: (2019)
Artesunate enhances adriamycin cytotoxicity by inhibiting glycolysis in adriamycin-resistant chronic myeloid leukemia K562/ADR cells
Authors: Chen, Li and Wang, Chao and Hu, Ning and Zhao, Hongmian
Journal: RSC Advances (2019): 1004--1014
Helicobacter pylori secreted protein HP1286 triggers apoptosis in macrophages via TNF-independent and ERK MAPK-dependent pathways
Authors: Tavares, Raquel and Pathak, Sushil Kumar
Journal: Frontiers in Cellular and Infection Microbiology (2017): 58
Promotion of osteointegration under diabetic conditions by tantalum coating-based surface modification on 3-dimensional printed porous titanium implants
Authors: Wang, Lin and Hu, Xiaofan and Ma, Xiangyu and Ma, Zhensheng and Zhang, Yang and Lu, Yizhao and Li, Xiang and Lei, Wei and Feng, Yafei
Journal: Colloids and Surfaces B: Biointerfaces (2016): 440--452
microRNA-186 inhibits cell proliferation and induces apoptosis in human esophageal squamous cell carcinoma by targeting SKP2
Authors: He, Wei and Feng, Jianfang and Zhang, Yan and Wang, Yuanyuan and Zang, Wenqiao and Zhao, Guoqiang
Journal: Laboratory Investigation (2016): 317--324
Hypoxia regulates TRAIL sensitivity of colorectal cancer cells through mitochondrial autophagy.
Authors: Knoll, Gertrud and Bittner, Sebastian and Kurz, Maria and Jantsch, Jonathan and Ehrenschwender, Martin
Journal: Oncotarget (2016)