Z-IETD-AFC *CAS 219138-02-0*

Important notes

It is important to store at <-15 °C and should be stored in cool, dark place.

It can be used within 12 months from the date of receipt. 

Following protocol only provides a guideline, and should be modified according to your specific needs.

General Solution Caspase Assays Using AMC, AFC, pNA, R110 and ProRed Substrates

1. Prepare a 10 mM stock solution in DMSO.
   
   
2. Prepare a 2X caspase substrate (50 µM) assay solution as the following: 50 µL substrate stock solution, 100 µL DTT (1M), 400 µL EDTA (100 mM), 10 mL Tris Buffer (20 mM), pH =7.4.
   
   
3. Mix equal volume of the caspase standards or samples with 2X caspase substrate assay solution, and incubate the solutions at room temperature for at least 1 hour.
   
   
4. Monitor the fluorescence using a fluorescence microplate reader, or absorbance using an absorbance microplate reader.

Cell Caspase Assays Using Cell-Permeable FMK Caspase Probes

1. Prepare a 2-5 mM stock solution in DMSO.
   
   
2. Treat cells as desired.
   
   
3. Prepare a 2X permeable caspase substrate (20 µM) assay solution by diluting the DMSO stock solution (from Step 2.1) in Hanks with 20 mM Hepes buffer (HHBS).
   
   
4. Mix equal volume of the treated cells with 2X caspase substrate assay solution (from Step 2.3), and incubate the cells in a 37°C, 5% CO₂ incubator for at least1 hour.
   
   
5. Wash the cells with HHBS for at least once.
   
   
6. Monitor the fluorescence intensity by a flow cytometer, a fluorescence microscope or a fluorescence microplate reader.

Cell Caspase Assays Using Cell-Permeable FMK Caspase Probes (For #13470-13476 only)

1. Prepare a 250X stock solution by adding 50 µL DMSO into the vial.
   
   
2. Treat cells as desired.
   
   
3. Add 250 X DMSO stock solution into the cell solution at a 1:250 ratio (such as 2 µL to 500 µL cells), and incubate the cells in a 37°C, 5% CO2 incubator for 1 hour.
   
   
4. Wash the cells with HHBS for at least once.
   
   
5. Monitor the fluorescence intensity by flow cytometer, fluorescence microscopy or fluorescent microplate reader.