Cell Meter™ Caspase 9 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 9 Activity Apoptosis Assay Kit *Blue Fluorescence*|
|Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Green Fluorescence*|
Fluorescence microplate reader
|Recommended plate||Black wall/clear bottom|
|Instrument specification(s)||Top or bottom read mode|
|Component A: Ac-LEHD-ProRed™||1 vial (50 µL, 200X)|
|Component B: Assay Buffer||1 bottle (10 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 9 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 WORKING SOLUTION
Add 5 µL of 200X Ac-LEHD-ProRed™ stock solution (Component A) into 1 mL of Assay Buffer (Component B) and mix well to make Caspase 9 Substrate working solution. Protect from light. Note: Caspase 9 working solution is not stable, use it promptly. 1 mL of Caspase 9 Substrate working solution is enough for 10 assays. Note: Aliquot and store unused caspase 9 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- 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-wel or 25 µL/well/384-well plate of Caspase 9 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-LEHD-CHO caspase 9 inhibitor to selected samples 10 minutes before adding Caspase 9 Substarte working solution at room temperature to confirm the inhibition of the caspase 9-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 non-adherent cells) at 800 rpm for 2 minutes (brake off) if using bottom read mode.
Open in Advanced Spectrum Viewer
|Name||Excitation (nm)||Emission (nm)||Extinction coefficient (cm -1 M -1)|
|Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Blue Fluorescence*||341||441||-|
|Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Green Fluorescence*||500||522||80000|
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