ApoSight™ Green Caspase 3/7 substrate

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
International	See distributors
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Custom size	Inquire
Shipping	Standard overnight for United States, inquire for international

Physical properties

Molecular weight	1290.61
Solvent	DMSO

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	80000
Excitation (nm)	500
Emission (nm)	522

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12171501

Overview SDS Protocol

Platform

Flow cytometer

Excitation	488 nm laser
Emission	530/30 nm filter
Instrument specification(s)	FITC channel

Fluorescence microscope

Excitation	FITC filter set
Emission	FITC filter set
Recommended plate	Black wall/clear bottom

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 for 96-well plate
Add equal volume of ApoSight™ Caspase 3/7 Substrate working solution
Incubate in a 5% CO₂ incubator at 37 °C for 60 minutes
Wash the cells 1-2 time with HHBS
Analyze the cells with flow cytometer with 530/30 nm filter (FITC channel) or with fluorescence microscope with FITC filter set

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 into the vial of ApoSight™ Green Caspase 3/7 Substrate to make 200X ApoSight™ Green Caspase 3/7 Substrate stock solution.
Note Aliquot in single use aliquots to avoid repeated freeze-thaw cycle. 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 Hanks and 20 mM Hepes buffer (HHBS, Cat# 20011) or buffer of your choice and 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.
Wash cells 1-2 times with HHBS or buffer of your choice.
Image with a fluorescence microscope using a FITC filter set.

Sample protocol for Flow Cytometry

Add 200 X ApoSight™ Green Caspase 3/7 Substrate stock solution into the cell solution at a 1:200 ratio, and incubate the cells in a 5% CO₂ incubator at 37 °C for 60 minutes.
Note The cells can be concentrated up to ~5 X 10⁶ cells/mL for ApoSight™ Green Caspase 3/7 Substrate labeling. The appropriate incubation time depends on the individual cell type and cell concentration used.
Monitor the fluorescence intensity with a flow cytometer using 530/30 nm filter (FITC channel).
Note To increase the signal/background ratio, spin down the cells at ~200 g for 5 minutes, wash cells with 1 mL washing buffer such as HHBS or buffer of your choice ones, and resuspend the cells in the desired amount of washing.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of ApoSight™ Green Caspase 3/7 substrate to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

	0.1 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	77.483 µL	387.414 µL	774.827 µL	3.874 mL	7.748 mL
5 mM	15.497 µL	77.483 µL	154.965 µL	774.827 µL	1.55 mL
10 mM	7.748 µL	38.741 µL	77.483 µL	387.414 µL	774.827 µL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	80000
Excitation (nm)	500
Emission (nm)	522

Images

Figure 1. The detection of caspase 3/7 activity in Jurkat cells with ApoSight™ Green Caspase 3/7 substrate. Jurkat cells (200,000 cells/well/ 96-well plate) were treated with 1 μM Staurosporine or DMSO for 4 hours. Cells were incubated with Caspase 3/7 Substrate working solution at 37°C for 1 hour. Images were taken with a fluorescence microscope using a FITC filter set.

References

View all 4 references: Citation Explorer

Cell culture conditions affect the ability of high content imaging assay to detect drug-induced changes in cellular parameters in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).
Authors: Balasubramanian, Bharathi and Belak, Vaclav and Verma, Isha and Prysiazhniuk, Yeva and Sannajust, Frederick and Trepakova, Elena S
Journal: Toxicology reports (2019): 305-320

In- and ex-vivo molecular imaging of apoptosis to assess sensitivity of non-small cell lung cancer to EGFR inhibitors using probe-based confocal laser endomicroscopy.
Authors: Guisier, Florian and Bohn, Pierre and Patout, Maxime and Piton, Nicolas and Farah, Insaf and Vera, Pierre and Thiberville, Luc and Salaün, Mathieu
Journal: PloS one (2017): e0180576

Polyphenols act synergistically with doxorubicin and etoposide in leukaemia cell lines.
Authors: Mahbub, A A and Le Maitre, C L and Haywood-Small, S L and Cross, N A and Jordan-Mahy, N
Journal: Cell death discovery (2015): 15043

Monitoring cleaved caspase-3 activity and apoptosis of immortalized oligodendroglial cells using live-cell imaging and cleaveable fluorogenic-dye substrates following potassium-induced membrane depolarization.
Authors: Smith, Graham S T and Voyer-Grant, Janine A M and Harauz, George
Journal: Journal of visualized experiments : JoVE (2012)