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Cell Meter™ No-Wash Live Cell Caspase 3/7 Activity Assay Kit
Blue 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™ U470, our recently developed cell-permeable fluorogenic caspase substrate, the first fluorogenic probe for the direct detection of caspase activities in live cells. ApoBrite U470 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™ U470 is much more robust, convenient and accurate. 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; U470 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; U470 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
20250200 Tests
Price
 
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200
Instrument settings
Flow cytometer
Excitation320 nm
Emission380 nm
Fluorescence microscope
Excitation320 nm
Emission380 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)DAPI channel
Fluorescence microplate reader
Excitation320 nm
Emission380 nm
Cutoff360 nm
Recommended plateSolid black
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Page updated on July 12, 2023