Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Green Fluorescence*
|Shipping||Standard overnight for United States, inquire for international|
|Extinction coefficient (cm -1 M -1)||80000|
|H-phrase||H303, H313, H333|
|Intended use||Research Use Only (RUO)|
|R-phrase||R20, R21, R22|
|Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Blue Fluorescence*|
|Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Red Fluorescence*|
|Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Green Fluorescence Optimized for Flow Cytometry*|
Extinction coefficient (cm -1 M -1)
Fluorescence microplate reader
|Recommended plate||Black wall/clear bottom|
|Instrument specification(s)||Top/Bottom read mode|
|Component A: Caspase 3/7 Substrate||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 3/7 Substrate working solution
- Incubate at room temperature for 1 hour
- Monitor fluorescence intensity at Ex/Em = 490/525 nm (Cutoff = 515 nm)
Thaw one vial of each kit component at room temperature before starting the experiment.
PREPARATION OF WORKING SOLUTION
Add 50 µL of Caspase 3/7 Substrate (Component A) into 10 mL of Assay Buffer (Component B) and mix well to make Caspase 3/7 Substrate working solution. Note: Aliquot and store the unused Caspase 3/7 Substrate (Components A) and Assay Buffer (Component B) at -20 oC. Avoid repeated freeze/thaw cycles
SAMPLE EXPERIMENTAL PROTOCOL
- For adherent cells: Plate cells overnight in growth medium at 20,000 cells/well/90 uL for a 96-well plate or 5,000cells/well/20mL for a 384-well plate.
- For non-adherent cells: Centrifuge the cells from the culture medium and then suspend the cell pellet in culture medium at 80,000 cells/well/90 uL for a 96-well poly-D lysine plate or 20,000 cells/well/20 uL for a 384-well poly-D lysine 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 (4 - 6 hours for Jurkat cells treated with camptothecin) to induce apoptosis.
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Caspase 3/7 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-DEVD-CHO caspase 3/7 inhibitor into selected samples 10 minutes before adding Caspase 3/7 working solution at room temperature to confirm the inhibition of the caspase 3/7-like activities.
- Centrifuge cell plate (especially for the non-adherent cells) at 800 rpm for 2 minutes (brake off).
- Monitor the fluorescence intensity with a fluorescence microplate reader at Ex/Em = 490/525 nm (Cutoff = 515 nm).
Open in Advanced Spectrum Viewer
|Extinction coefficient (cm -1 M -1)||80000|
|Name||Excitation (nm)||Emission (nm)|
|Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Blue Fluorescence*||341||441|
|Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Red Fluorescence*||532||619|
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