Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Blue Fluorescence*
|Shipping||Standard overnight for United States, inquire for international|
|H-phrase||H303, H313, H333|
|Intended use||Research Use Only (RUO)|
|R-phrase||R20, R21, R22|
|Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Green Fluorescence*|
|Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Red Fluorescence*|
Fluorescence microplate reader
|Recommended plate||Black wall/clear bottom|
|Instrument specification(s)||Top/Bottom read mode|
|Component A: Caspase 8 Substrate (200X Stock Solution)||2 vials (50 µL/vial)|
|Component B: Assay Buffer||1 bottle (20 mL)|
AT A GLANCE
- Prepare cells with test compounds (100 µL/well/96-well plate or 25 µL/well/384-well plate)
- Add equal volume of Caspase 8 Substrate working solution (100 µL/well/96-well plate or 25 µL/well/384-well plate)
- Incubate at room temperature for 30 - 60 minutes
- Monitor fluorescence increase at Ex/Em = 370/450 nm (Cutoff = 420 nm)
Thaw all the kit components at room temperature before starting the experiment.
PREPARATION OF WORKING SOLUTION
Add 50 µL of Caspase 8 Substrate (Component A) into 10 mL of Assay Buffer (Component B) and mix well to make Caspase 8 Substrate working solution. Protect from light. Note: Caspase 8 Substrate working solution is not stable, use it promptly. Note: Aliquot and store unused Caspase 8 Substrate (Component A) and Assay Buffer (Component B) at -20 oC. Avoid repeated freeze/thaw cycles.
For guidelines on cell sample preparation, please visit
SAMPLE EXPERIMENTAL PROTOCOL
- Treat cells by adding 10 µL/well of 10X test compounds (96-well plate) or 5 µL/well of 5X test compounds (384-well plate) into PBS or the desired buffer. For blank wells (medium without the cells), add the same amount of compound buffer.
- Incubate the cell plate in a 5% CO2, 37°C incubator for a desired period of time (4 - 6 hours for Jurkat cells treated with staurosporine) to induce apoptosis.
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Caspase 8 Substrate working solution.
- Incubate the Caspase 8 Substrate working solution plate at room temperature for 30 to 60 minutes, protected from light. Note: If desired, add 1 µL of the 1 mM Ac-IETD-CHO caspase 8 inhibitor to selected samples 10 minutes before adding Caspase 8 Substrate working solution at room temperature to confirm the inhibition of the caspase 8-like activities.
- Centrifuge cell plate (especially for the non-adherent cells) at 800 rpm for 2 minutes (brake off).
- Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 370/450 nm (Cutoff = 420 nm).
Open in Advanced Spectrum Viewer
|Name||Excitation (nm)||Emission (nm)||Extinction coefficient (cm -1 M -1)|
|Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Green Fluorescence*||500||522||80000|
|Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Red Fluorescence*||532||619||-|
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