Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Red 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 *Blue Fluorescence*|
|Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Green Fluorescence*|
Fluorescence microplate reader
|Recommended plate||Black wall/clear bottom|
|Instrument specification(s)||Top or bottom read mode|
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 1 hour
- Monitor fluorescence intensity (top or bottom read mode) at Ex/Em = 540/620 nm (Cutoff = 610 nm)
Thaw all the kit components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. Ac-IETD-ProRed™ stock solution (200X):
Add 65 µL DMSO into the vial of Ac-IETD-ProRed™ (Component A) and mix well to make 200X Ac-IETD-ProRed™ stock solution. Protect from light.
PREPARATION OF WORKING SOLUTION
Add 50 µL of 200X Ac-IETD-ProRed™ stock solution into 10 mL of Assay Buffer (Component B) and mix well to make Caspase 8 Substrate working solution. Protect from light. Note: Caspase 8 working solution is not stable, use it promptly.
SAMPLE EXPERIMENTAL PROTOCOL
- For adherent cells: Plate cells overnight in growth medium at 20,000 cells/well/90 uL for a 96-well or 5,000cells/well/20 uL for a 384-well plate black wall/clear bottom plate.
- For non-adherent cells: Centrifuge the cells from the culture medium and then suspend the cell pellet in culture medium at 80,000 to 200,000 cells/well/90 uL for a 96-well or 20,000 to 50,000 cells/well/20 uL for a 384-well black wall/clear bottom plate. Centrifuge the plate at 800 rpm for 2 minutes with brake off prior to the experiments. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for apoptosis induction.
- 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 37°C, 5% CO2, incubator for a desired period of time (3 - 4 hours for Jurkat cells treated with staurosporine) to induce apoptosis.
- Add 100 µL/well/96-well or 25 µL/well/384-well plate of Caspase 8 Substrate working solution.
- Incubate the plate at room temperature for at least 1 hour, 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.
- Monitor the fluorescence intensity with a fluorescence microplate reader (either top or bottom read mode) at Ex/Em = 540/620 nm (Cutoff = 610 nm). Note: Sometimes, bottom read gives better signal to background ratio, centrifuge cell plate (especially for the nonadherent cells) at 800 rpm for 2 minutes (brake off) if using bottom read mode.
|Name||Excitation (nm)||Emission (nm)||Extinction coefficient (cm -1 M -1)|
|Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Blue Fluorescence*||341||441||-|
|Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Green Fluorescence*||500||522||80000|
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