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Cell Meter™ Multiplexing Caspase 3/7, 8 and 9 Activity Assay Kit *Triple Fluorescence Colors*

Detection of Caspase Activities in Jurkat cells. Jurkat cells were seeded on the same day at 200,000 cells/well in a Costar black wall/clear bottom 96-well plate. The cells were treated with staurosporine at the final concentration of 1 mM for 4 hours (Red Bar) while the untreated cells were used as control (Blue Bar). The single-caspase assay loading solution (100 uL/well) was added (in #1 for caspase 3/7, #2 for caspase 8 or #3 for caspase 9) or Triple-caspase assay loading solution (#4 for caspase 3/7, 8 and 9 together) was added, and incubated at room temperature for 1 hour. The fluorescence intensity was measured with FlexStation fluorescence microplate reader at the indicated wavelength. The caspase 3/7, 8 and 9 activities can be detected in a single assay without interferences from other caspases.
Detection of Caspase Activities in Jurkat cells. Jurkat cells were seeded on the same day at 200,000 cells/well in a Costar black wall/clear bottom 96-well plate. The cells were treated with staurosporine at the final concentration of 1 mM for 4 hours (Red Bar) while the untreated cells were used as control (Blue Bar). The single-caspase assay loading solution (100 uL/well) was added (in #1 for caspase 3/7, #2 for caspase 8 or #3 for caspase 9) or Triple-caspase assay loading solution (#4 for caspase 3/7, 8 and 9 together) was added, and incubated at room temperature for 1 hour. The fluorescence intensity was measured with FlexStation fluorescence microplate reader at the indicated wavelength. The caspase 3/7, 8 and 9 activities can be detected in a single assay without interferences from other caspases.
<strong>cSBL induced apoptosis in H2452 and MSTO cells via activation of the caspase pathway. </strong>Caspase-3, -8, and -9 activation was detected by western blotting (A, B) or fluorometry (C, D). Fluorometry was performed independently three times and data are expressed as the mean ± SD. The statistical significance of these experiments compared with the control is shown in as follows: *P<0.05, **P<0.01, ***P<0.001. Source: <strong>Sialic acid-binding lectin from bullfrog eggs inhibits human malignant mesothelioma cell growth <em>in vitro</em> and <em>in vivo</em></strong> by Tatsuta T et al., <em>PLOS</em>, Jan. 2018.
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Catalog Number22820
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Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
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ShippingStandard overnight for United States, inquire for international
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


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. Caspases activation is widely accepted as a reliable indicator for cell apoptosis. This particular kit is designed to simultaneously monitor four key caspases (caspase-3/7, 8 and 9) activation involved in cell apoptosis using three distinct fluorescent colors. This kit uses DEVD-ProRed™, IETD-R110 and LEHD-AMC as fluorogenic indicators for caspase 3/7, 8 and 9 activity respectively. Upon caspase cleavages, DEVD-ProRed™, IETD-R110 and LEHD-AMC caspase substrates generate three distinct fluorophores: ProRed™ (red fluorescence), R110 (green fluorescence) and AMC (blue fluorescence), which can be readily monitored in a single assay due to their nice spectral separation.

Platform


Fluorescence microplate reader

ExcitationSee Table 1
EmissionSee Table 1
Recommended plateBlack wall/clear bottom
Instrument specification(s)Use either top or bottom read mode

Components


Component A: Caspase 3/7 Substrate (DEVD-ProRed™, 200X )1 vial (50 µL/vial)
Component B: Caspase 8 Substrate (IETD-R110, 200X)1 vial (50 µL/vial)
Component C: Caspase 9 Substrate (LEHD-AMC, 200X)1 vial (50 µL/vial)
Component D: Assay Buffer1 bottle (30 mL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells with test compounds
  2. Add equal volume of caspase working solution
  3. Incubate at room temperature for 30 min to 1 hour
  4. Monitor fluorescence intensity

Important notes
Thaw kit components at room temperature before use.

PREPARATION OF WORKING SOLUTION

1. Single caspase activity working solution
Make caspase 3/7, caspase 8 or caspase 9 working solution by adding 50 µL of substrate (Component A, B or C) into 10 mL of Assay Buffer (Component D), and mix them well.

2. Dual- or tri- caspase activity working solution:
Add 50 µL of each interested caspase substrate into 10 mL of Assay Buffer (Component D) together to make the working solution. Note: Please prepare the tested substrate solutions and the needed volume proportionally.

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-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 - 5 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 desired caspase assay working solution.

  4. Incubate the plate at room temperature for at least 30 to 60 min, protected from light. Note: If desired, add 1 µL of 1 mM caspase inhibitor to selected samples 10 minutes before adding the assay loading solution at room temperature to confirm the inhibition of caspase activities.

  5. Monitor the fluorescence intensity as indicated in the table with either top or bottom read mode. Note: Sometimes, bottom read gives better signal to background ratio, centrifuge cell plate (especially for the nonadherent cells) at 800 rpm for 2 minutes (brake off) if using bottom read mode.

Table 1. Spectral wavelengths for Caspases 3/7, 8, and 9.

Capase Fluorescence Excitation Emission
Caspase 3/7 Red 535 nm 620 nm
Caspase 8 Green 490 nm 525 nm
Caspase 9 Blue 370 nm 450 nm

 

Citations


View all 23 citations: Citation Explorer
Asparagus officinalis combined with paclitaxel exhibited synergistic anti-tumor activity in paclitaxel-sensitive and-resistant ovarian cancer cells
Authors: Zhang, Xin and Wang, Jiandong and Fan, Yali and Zhao, Ziyi and Paraghamian, Sarah E and Hawkins, Gabrielle M and Buckingham, Lindsey and O’Donnell, Jillian and Hao, Tianran and Suo, Hongyan and others,
Journal: Journal of Cancer Research and Clinical Oncology (2022): 1--13
Changes in Apoptotic Pathways in MOLM-13 Cell Lines after Induction of Resistance to Hypomethylating Agents
Authors: Janotka, L'ubo{\v{s}} and Messingerov{\'a}, Lucia and {\v{S}}imoni{\v{c}}ov{\'a}, Krist{\'\i}na and Kavcov{\'a}, Helena and Elefantov{\'a}, Katar{\'\i}na and Sulov{\'a}, Zdena and Breier, Albert
Journal: International journal of molecular sciences (2021): 2076
Hypertonicity-enforced BCL-2 addiction unleashes the cytotoxic potential of death receptors
Authors: Sirtl, Simon and Knoll, Gertrud and Trinh, Dieu Thuy and Lang, Isabell and Siegmund, Daniela and Gross, Stefanie and Schuler-Thurner, Beatrice and Neubert, Patrick and Jantsch, Jonathan and Wajant, Harald and others,
Journal: Oncogene (2018): 4122--4136
Hypertonicity-imposed BCL-XL addiction primes colorectal cancer cells for death
Authors: Heimer, Sina and Knoll, Gertrud and Steixner, Charlotte and Calance, Diana Nicoleta and Trinh, Dieu Thuy and Ehrenschwender, Martin
Journal: Cancer Letters (2018)
Sialic acid-binding lectin from bullfrog eggs inhibits human malignant mesothelioma cell growth in vitro and in vivo
Authors: Tatsuta, Takeo and Satoh, Toshiyuki and Sugawara, Shigeki and Hara, Akiyoshi and Hosono, Masahiro
Journal: PloS one (2018): e0190653
Role of lincRNA-p21 in the protective effect of macrophage inhibition factor against hypoxia/serum deprivation-induced apoptosis in mesenchymal stem cells
Authors: Xia, Wenzheng and Zhuang, Lei and Hou, Meng
Journal: International journal of molecular medicine (2018)
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
Hyperosmotic stress enhances cytotoxicity of SMAC mimetics
Authors: Bittner, Sebastian and Knoll, Gertrud and Ehrenschwender, Martin
Journal: Cell death \& disease (2017): e2967--e2967
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