Cell Meter™ Live Cell Caspase 3/7 Imaging Kit Green Fluorescence

Cell Meter™ Live Cell No Wash Caspase 3/7 Imaging Kit uses our recently developed cell-permeable fluorogenic caspase substrate, ApoSight™ Green Caspase 3/7, the first fluorogenic probe, for the direct detection of caspase activities in live cells. ApoSight™ Green Caspase 3/7 substrate consists of three moieties including a). a masked fluorophore, b). a caspase-selective peptide fragment (DEVD), and c). a 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, ApoSight™ Green Caspase 3/7 substrate is much more robust, convenient, and accurate. ApoSight™ Green Caspase 3/7 substrate releases a fluorophore that has Ex/Em ~490/520 nm. 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 irreversible 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 extremely low throughput; d). FMK peptides are not stable in aqueous solutions and must be used immediately. Cell Meter™ Live Cell No Wash Caspase 3/7 Imaging Kit provides an optimized fluorescence imaging assay for monitoring caspase3/7 activity. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation.

Example protocol

AT A GLANCE

Protocol summary

Prepare cells with test compounds at a density of 5 × 10⁴ to 2 × 10⁵ cells/100 µL/well/96-well plate
Add equal volume of Caspase 3/7 Substrate working solution
Incubate in a 5% CO2 incubator at 37 °C for 60 minutes
Image with a fluorescence microscope using a FITC filter set

Important

Thaw all the kit components at room temperature before use.

CELL PREPARATION

For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html

PREPARATION OF STOCK SOLUTIONS

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

ApoSight™ Green Caspase 3/7 Substrate stock solution(200X)

Add 50 µL of DMSO (Component C) into the vial of ApoSight™ Green Caspase 3/7 Substrate (Component A) to make 200X ApoSight™ Green Caspase 3/7 Substrate stock solution.
Note Aliquot in smaller vials to avoid repeated freeze thaw cycles. Protect from light.

PREPARATION OF WORKING SOLUTION

ApoSight™ Green Caspase 3/7 Substrate working solution

Prepare ApoSight™ Green Caspase 3/7 substrate working solution by mixing 5 µL of 200X ApoSight™ Green Caspase 3/7 Substrate stock solution with 1 mL of assay buffer (Component B). Mix well.
Note 100 µL of ApoSight™ Green Caspase 3/7 substrate working solution is enough for 10 tests in a 96-well plate format
Note Prepare enough ApoSight™ Green Caspase 3/7 substrate working solution right before the experiment, and use promptly.

SAMPLE EXPERIMENTAL PROTOCOL

Examples for inducing apoptosis in suspension culture

Treat Jurkat cells with 2 μg/mL camptothecin for 3 hours

Treat Jurkat cells with 1 μM staurosporine for 3-4 hours

Treat HL-60 cells with 4 μg/mL camptothecin for 4 hours

Treat HL-60 cells with 1 μM staurosporine for 4 hours.
Note Each cell line should be evaluated on an individual basis to determine the optimal cell density for apoptosis induction.

Sample protocol for Fluorescence Microscopy

Prepare cells with test compounds at a density of 5 × 10⁴ to 2 × 10⁵ cells/100 µL/well/96-well plate.
Add equal volume of Caspase 3/7 Substrate working solution to the cells (100 µL/well/96 well-plate).
Incubate in a 5% CO₂ incubator at 37 °C for 60 minutes.
Image with a fluorescence microscope using a FITC filter set.

Spectrum

Open in Advanced Spectrum Viewer

Citations

View all 1 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

References

View all 19 references: Citation Explorer

68Ga-Galmydar: A PET imaging tracer for noninvasive detection of Doxorubicin-induced cardiotoxicity.
Authors: Sivapackiam, Jothilingam and Kabra, Shivesh and Speidel, Sylvia and Sharma, Monica and Laforest, Richard and Salter, Amber and Rettig, Michael P and Sharma, Vijay
Journal: PloS one (2019): e0215579

Repurposing the Electron Transfer Reactant Phenazine Methosulfate (PMS) for the Apoptotic Elimination of Malignant Melanoma Cells through Induction of Lethal Oxidative and Mitochondriotoxic Stress.
Authors: Hua, Anh B and Justiniano, Rebecca and Perer, Jessica and Park, Sophia L and Li, Hui and Cabello, Christopher M and Wondrak, Georg T
Journal: Cancers (2019)

Salt-Inducible Kinase 1 (SIK1) is Induced by Alcohol and Suppresses Microglia Inflammation via NF-κB Signaling.
Authors: Zhang, Yu and Gao, Weida and Yang, Kongbin and Tao, Haiquan and Yang, Haicheng
Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology (2018): 1411-1421

C10ORF10/DEPP-mediated ROS accumulation is a critical modulator of FOXO3-induced autophagy.
Authors: Salcher, S and Hermann, M and Kiechl-Kohlendorfer, U and Ausserlechner, M J and Obexer, P
Journal: Molecular cancer (2017): 95

A fluorescent light-up aggregation-induced emission probe for screening gefitinib-sensitive non-small cell lung carcinoma.
Authors: Hu, Yi and Shi, Leilei and Su, Yue and Zhang, Chuan and Jin, Xin and Zhu, Xinyuan
Journal: Biomaterials science (2017): 792-799

Page updated on October 24, 2024

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