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Cell Meter™ No-Wash Live Cell Caspase 3/7 Activity Assay Kit
Red Fluorescence
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.
<p>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. &nbsp;ApoBrite&trade; V570 caspase 3/7 dye loading solution was then added to the well. After incubation for 2 h at 37 &deg;C, the cells were washed once with HHBS buffer and treated with staurosporine (1 &mu;M) apoptosis inducer for 1 hr. The images were acquired using a fluorescence microscope equipped with DAPI filter set.</p>
<p>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. &nbsp;ApoBrite&trade; V570 caspase 3/7 dye loading solution was then added to the well. After incubation for 2 h at 37 &deg;C, the cells were washed once with HHBS buffer and treated with staurosporine (1 &mu;M) apoptosis inducer for 1 hr. The images were acquired using a fluorescence microscope equipped with DAPI filter set.</p>
protocol
Protocol
sds
SDS
COA
CatalogSize
Price
Quantity
20260200 Tests
Price
 
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
Instrument settings
Flow cytometer
Excitation405 nm
Emission570 nm
Instrument specification(s)FL3 channel for 7-AAD staining
Fluorescence microscope
ExcitationDAPI channel
EmissionDAPI channel
Recommended plateBlack wall/clear bottom
Instrument specification(s)TRITC channel for 7-AAD staining
Fluorescence microplate reader
Excitation405 nm
Emission570 nm
Cutoff540 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode
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Page updated on February 1, 2023