Cell Meter™ No-Wash Live Cell Caspase 3/7 Activity Assay Kit *Red Fluorescence*
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Additional ordering information
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Storage, safety and handling
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Related products
Overview | ![]() ![]() |
The activation of caspase 3/7 is important for the initiation of apoptosis. Our Cell Meter™ No-Wash Live Cell Caspase Activity Assay Kits are based on ApoBrite™ V600, our recently developed cell-permeable fluorogenic caspase substrate, the first fluorogenic probe for the direct detection of caspase activities in live cells. ApoBrite V600 consists of three moieties including a). masked fluorophore, b). caspase-selective peptide fragment (DEVD), and c). cell-penetrating moiety. The cell-penetrating moiety carries the probe into live cells. Upon entering live cells the caspase-selective peptide fragment is cleaved by a caspase to release the masked fluorophore. The intensity of recovered fluorescence is directly related to the activity of caspase to be measured. Compared to the existing caspase assays in live cells, ApoBrite™ V600 is much more robust, convenient and accurate. ApoBrite™ V600 releases a fluorophore that has a large Stokes shift, and can be well excited with violet laser that is installed most of new flow cytometers. It does not need a DNA interaction to be fluorescent as reported for NucView reagents. It does not inhibit caspase activity as reported for the FMK peptide probes. Although fluorescent FMK peptide inhibitors of caspases are widely used for detecting caspase activities in live cells, this technology has a few severe limitations: a). FMK caspase inhibitors have high cytotoxicity since FMK peptides bind covalently to active caspases; b). The irreversibly covalent binding of FMK peptides to caspases inhibits caspase activities, causing false positive apoptosis; c). FMK assays have extremely high background, and require intensive washings, resulting in very low through put; d). FMK peptides are not stable in aqueous solutions, and have to be used immediately.
Platform
Flow cytometer
Excitation | 405 nm |
Emission | 570 nm |
Instrument specification(s) | FL3 channel for 7-AAD staining |
Fluorescence microscope
Excitation | DAPI channel |
Emission | DAPI channel |
Recommended plate | Black wall/clear bottom |
Instrument specification(s) | TRITC channel for 7-AAD staining |
Fluorescence microplate reader
Excitation | 405 nm |
Emission | 570 nm |
Cutoff | 540 nm |
Recommended plate | Black wall/clear bottom |
Instrument specification(s) | Bottom read mode |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare cells
- Remove the growth medium, wash cells
- Incubate Caspase 3/7 Substrate working solution at 37 ºC for 2 hours
- Wash cells and replace with growth medium
- Induce apoptosis
- Monitor fluorescence intensity (bottom read mode) at Ex/Em = 405/570 nm (Cutoff = 540 nm), fluorescence microscope with DAPI filter, or flow cytometer with Ex/Em = 405/470 nm filters
Important notes
Thaw all the components at room temperature before starting the experiment.
PREPARATION OF WORKING SOLUTION
Add 2 μL of 500X ApoBrite™ V570 Caspase 3/7 Substrate stock solution (Component A) into 1 mL HHBS (Component B) to make 1X ApoBrite™ V570 Caspase 3/7 Substrate working solution.
For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
SAMPLE EXPERIMENTAL PROTOCOL
- Culture cells to a density optimal for apoptosis induction according to your specific induction protocol, but not to exceed 2 x 106 cells/mL for suspension cells. For adherent cells, plate cells at 30,000 - 80,000 cells/well/96-well plate overnight.
- Remove growth medium, and wash once with HHBS (Component B) or buffer of your choice.
- Add 100 µL/well/96 well plate of 1X ApoBrite™ V570 Caspase 3/7 Substrate working solution, and incubate at 37 ºC, 5% CO2 incubator for 2 hours.
- Remove 1X ApoBrite™ V570 Caspase 3/7 Substrate working solution and wash once with HHBS (Component B).
- Add growth medium back to the ApoBrite™ V570 Caspase 3/7 loaded cells, and treat the cells as desired for apoptosis. Here are a few examples for inducing apoptosis in cell culture:
- Treating Jurkat cells with 2 µg/ml camptothecin for 3 hours.
- Treating Jurkat cells with 1 µM staurosporine for 3 hours. Hela cells for 1 hour.
- Treating HL-60 cells with 4 µg/ml camptothecin for 4 hours.
- Treating HL-60 cells with 1 µM staurosporine for 4 hours.
- Treating Hela cells with 1 µM staurosporine for 1 hour. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for apoptosis induction.
- Treating Jurkat cells with 2 µg/ml camptothecin for 3 hours.
- If desired, label the cells with a DNA stain (such as 7-AAD for dead cells).
- Monitor the fluorescence intensity by fluorescence microscopy, flow cytometer, or fluorescence microplate reader at Ex/Em = 405/570 nm (for 7-AAD, Ex/Em = 535/650 nm).
For flow cytometry (suspension cells): Monitor the fluorescence intensity using Ex/Em = 405/570 nm filters (FL3 channel for 7-AAD staining). Gate on the cells of interest, excluding debris.
For fluorescence microscope: Observe cells under a fluorescence microscope using Ex/Em = 405/570 nm or DAPI channel (TRITC channel for 7-AAD staining).
For fluorescence microplate reader: Monitor the fluorescence intensity (bottom read mode) with a fluorescence plate reader at Ex/Em = 405/570 nm (Cutoff = 540 nm).
Images

Figure 1. The fluorescence microscope images of normal Hela cells (A) and apoptotic Hela cells (B). Hela cells were cultured in a 96-well plate, and washed twice with HHBS buffer. ApoBrite™ V570 caspase 3/7 dye loading solution was then added to the well. After incubation for 2 h at 37 °C, the cells were washed once with HHBS buffer and treated with staurosporine (1 μM) apoptosis inducer for 1 hr. The images were acquired using a fluorescence microscope equipped with DAPI filter set.
Citations
View all 15 citations: Citation Explorer
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
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
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
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)
Authors: Knoll, Gertrud and Bittner, Sebastian and Kurz, Maria and Jantsch, Jonathan and Ehrenschwender, Martin
Journal: Oncotarget (2016)
Role of delta-like ligand-4 in chemoresistance against docetaxel in MCF-7 cells
Authors: Wang, Q and Shi, Y and Butler, HJ and Xue, J and Wang, G and Duan, P and Zheng, H
Journal: Human & Experimental Toxicology (2016): 0960327116650006
Authors: Wang, Q and Shi, Y and Butler, HJ and Xue, J and Wang, G and Duan, P and Zheng, H
Journal: Human & Experimental Toxicology (2016): 0960327116650006
Angiopoietins Modulate Survival, Migration, and the Components of the Ang-Tie2 Pathway of Chronic Lymphocytic Leukaemia (CLL) Cells In Vitro
Authors: Palma, Luis Mario Aguirre and Flamme, Hanna and Gerke, Iris and Kreuzer, Karl-Anton
Journal: Cancer Microenvironment (2016): 13--26
Authors: Palma, Luis Mario Aguirre and Flamme, Hanna and Gerke, Iris and Kreuzer, Karl-Anton
Journal: Cancer Microenvironment (2016): 13--26
t-BHQ Provides Protection against Lead Neurotoxicity via Nrf2/HO-1 Pathway
Authors: Ye, Fang and Li, Xiaoyi and Li, Lili and Yuan, Jing and Chen, Jun
Journal: Oxidative medicine and cellular longevity (2015)
Authors: Ye, Fang and Li, Xiaoyi and Li, Lili and Yuan, Jing and Chen, Jun
Journal: Oxidative medicine and cellular longevity (2015)
Insulin improves osteogenesis of titanium implants under diabetic conditions by inhibiting reactive oxygen species overproduction via the PI3K-Akt pathway
Authors: Wang, Lin and Zhao, Xiong and Wei, Bo-yuan and Liu, Yi and Ma, Xiang-yu and Wang, Jian and Cao, Peng-chong and Zhang, Yang and Yan, Ya-bo and Lei, Wei and others, undefined
Journal: Biochimie (2015): 85--93
Authors: Wang, Lin and Zhao, Xiong and Wei, Bo-yuan and Liu, Yi and Ma, Xiang-yu and Wang, Jian and Cao, Peng-chong and Zhang, Yang and Yan, Ya-bo and Lei, Wei and others, undefined
Journal: Biochimie (2015): 85--93
Glucose promotes cell proliferation, glucose uptake and invasion in endometrial cancer cells via AMPK/mTOR/S6 and MAPK signaling
Authors: Han, Jianjun and Zhang, Lu and Guo, Hui and Wysham, Weiya Z and Roque, Dario R and Willson, Adam K and Sheng, Xiugui and Zhou, Chunxiao and Bae-Jump, Victoria L
Journal: Gynecologic oncology (2015): 668--675
Authors: Han, Jianjun and Zhang, Lu and Guo, Hui and Wysham, Weiya Z and Roque, Dario R and Willson, Adam K and Sheng, Xiugui and Zhou, Chunxiao and Bae-Jump, Victoria L
Journal: Gynecologic oncology (2015): 668--675
IL-7 abrogates the immunosuppressive function of human double-negative T cells by activating Akt/mTOR signaling
Authors: Allgäuer, Andrea and Schreiner, Elisabeth and Ferrazzi, Fulvia and Ekici, Arif B and Gerbitz, Armin and Mackensen, Andreas and Völkl, Simon
Journal: The Journal of Immunology (2015): 3139--3148
Authors: Allgäuer, Andrea and Schreiner, Elisabeth and Ferrazzi, Fulvia and Ekici, Arif B and Gerbitz, Armin and Mackensen, Andreas and Völkl, Simon
Journal: The Journal of Immunology (2015): 3139--3148