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Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Blue Fluorescence*

Our Cell Meter™ assay kits are a set of tools for monitoring cellular functions. There are a variety of parameters that can be used to monitor cell apoptosis. This particular kit is designed to monitor cell apoptosis by measuring caspase 8 activity. Caspase 8 is a caspase protein, encoded by the CASP8 gene. Caspase 8 also plays an important role in neurodegenerative diseases such as Huntington disease. Caspase 8 is proven to have substrate selectivity for the peptide sequence Ile-Glu-Thr-Asp (IETD). This kit uses (Ac-IETD)-AMC as a fluorogenic indicator for caspase 8 activity. Cleavage of AMC peptides by caspase 8 generates strongly fluorescent AMC. This spectral feature makes the kit compatible with the DAPI filter set. The kit provides all the essential components with an optimized assay protocol. The assay can be readily adapted for high throughput screenings. It can be used to either quantify the activated caspase 8 activities in apoptotic cells or screen the caspase 8 inhibitors.

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 8 Substrate working solution (100 µL/well/96-well plate or 25 µL/well/384-well plate)
  3. Incubate at room temperature for 30 - 60 minutes
  4. Monitor fluorescence increase at Ex/Em = 370/450 nm (Cutoff = 420 nm)

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

PREPARATION OF WORKING SOLUTION

Add 50 µL of Caspase 8 Substrate (Component A) into 10 mL of Assay Buffer (Component B) and mix well to make Caspase 8 Substrate working solution. Protect from light. Note: Caspase 8 Substrate working solution is not stable, use it promptly. Note: Aliquot and store unused Caspase 8 Substrate (Component A) and Assay Buffer (Component 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

  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 5% CO2, 37°C incubator for a desired period of time (4 - 6 hours for Jurkat cells treated with staurosporine) to induce apoptosis.

  3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Caspase 8 Substrate working solution.

  4. Incubate the Caspase 8 Substrate working solution plate at room temperature for 30 to 60 minutes, protected from light. Note: If desired, add 1 µL of the 1 mM Ac-IETD-CHO caspase 8 inhibitor to selected samples 10 minutes before adding Caspase 8 Substrate working solution at room temperature to confirm the inhibition of the caspase 8-like activities.

  5. Centrifuge cell plate (especially for the non-adherent cells) at 800 rpm for 2 minutes (brake off).

  6. Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 370/450 nm (Cutoff = 420 nm).

Spectrum

Product family

Citations

View all 20 citations: Citation Explorer
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
Contact-dependent signaling triggers tumor-like proliferation of CCM3 knockout endothelial cells in co-culture with wild-type cells
Authors: Rath, Matthias and Schwefel, Konrad and Malinverno, Matteo and Skowronek, Dariush and Leopoldi, Alexandra and Pilz, Robin A and Biedenweg, Doreen and Bekeschus, Sander and Penninger, Josef M and Dejana, Elisabetta and others,
Journal: Cellular and Molecular Life Sciences (2022): 1--20
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)
Role of endothelial microRNA-150 in pulmonary arterial hypertension
Authors: Russomanno, Giusy and Jo, Kyeong Beom and Abdul-Salam, Vahitha B and Morgan, Claire and Alzaydi, Mai and Wilkins, Martin R and Wojciak-Stothard, Beata
Journal: bioRxiv (2020)
RPL21 siRNA blocks proliferation in pancreatic cancer cells by inhibiting DNA replication and inducing G1 arrest and apoptosis
Authors: Li, Chaodong and Ge, Mei and Chen, Daijie and Sun, Tao and Jiang, Hua and Xie, Yueqing and Lu, Huili and Zhang, Baohong and Han, Lei and Chen, Junsheng and others,
Journal: Frontiers in oncology (2020): 1730
Page updated on December 6, 2024

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Catalog Number22812
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Spectral properties

Excitation (nm)

341

Emission (nm)

441

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

Platform

Fluorescence microplate reader

Excitation370 nm
Emission450 nm
Cutoff420 nm
Recommended plateBlack wall, clear bottom
Instrument specification(s)Top, Bottom read mode

Components

Detection of caspase 8 activity in Jurkat cells using Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Blue Fluorescence*. 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 8 working solution (100 µL/well) was added and incubated at room temperature for 30 minutes. The fluorescence intensity was measured at Ex/Em = 370/450 nm (Cutoff = 420 nm) with a FlexStation™ microplate reader (Molecular Devices).
Detection of caspase 8 activity in Jurkat cells using Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Blue Fluorescence*. 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 8 working solution (100 µL/well) was added and incubated at room temperature for 30 minutes. The fluorescence intensity was measured at Ex/Em = 370/450 nm (Cutoff = 420 nm) with a FlexStation™ microplate reader (Molecular Devices).
Detection of caspase 8 activity in Jurkat cells using Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Blue Fluorescence*. 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 8 working solution (100 µL/well) was added and incubated at room temperature for 30 minutes. The fluorescence intensity was measured at Ex/Em = 370/450 nm (Cutoff = 420 nm) with a FlexStation™ microplate reader (Molecular Devices).