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.

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
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H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12352200

Overview SDS Protocol

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

Excitation	See Table 1
Emission	See Table 1
Recommended plate	Black wall/clear bottom
Instrument specification(s)	Use either top or bottom read mode

Components

Example protocol

AT A GLANCE

Protocol summary

Prepare cells with test compounds
Add equal volume of caspase working solution
Incubate at room temperature for 30 min to 1 hour
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

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.
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.
Add 100 µL/well/96-well or 25 µL/well/384-well plate of desired caspase assay working solution.
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.
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

Images

Figure 1. 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.

Figure 2. cSBL induced apoptosis in H2452 and MSTO cells via activation of the caspase pathway. 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: Sialic acid-binding lectin from bullfrog eggs inhibits human malignant mesothelioma cell growth in vitro and in vivo by Tatsuta T et al., PLOS, Jan. 2018.

Citations

View all 24 citations: Citation Explorer

Microalga Chlorella sp. extract induced apoptotic cell death of cholangiocarcinoma via AKT/mTOR signaling pathway
Authors: Sawasdee, Nunghathai and Jantakee, Kanyaluck and Wathikthinnakon, Methi and Panwong, Suthida and Pekkoh, Jeeraporn and Duangjan, Kritsana and Yenchitsomanus, Pa-thai and Panya, Aussara
Journal: Biomedicine \& Pharmacotherapy (2023): 114306

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

An{\'a}lise da estrutura tridimensional, especificidade e atividade antic{\^a}ncer da lectina Canavalia villosa (Benth)
Authors: L{\'o}ssio, Cl{\'a}udia Figueiredo
Journal: (2021)

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