Z-IETD-AFC is a fluorescent substrate for caspase-8 and granzyme B. It can be used to quantify cysteinyl aspartate-8 or granzyme B activity.

Instructions
a. Solution preparation:
1. Prepare Z-IETD-AFC powder, dissolve it in DMSO, prepare a 10 mM stock solution, store below - 15°C, and protect from light. Prepare DTT (1 M), EDTA (100 mM), and Tris buffer (20 mM, pH=7.4).
2. Mix 50 μL Z-IETD-AFC stock solution, 100 μL DTT, 400 μL EDTA, and 10 mL Tris buffer to prepare a 2x Caspase substrate detection solution, in which the final concentration of Z-IETD-AFC is 50 μM.
b. Operation steps:
1. Sample processing: Mix samples or cell lysates containing Caspase-8 activity with an equal volume of 2x Caspase substrate detection solution and incubate at room temperature for at least 1 hour.
2. Detection and analysis: The fluorescence intensity was monitored using a fluorescence microplate reader with an excitation wavelength of 376 nm and an emission wavelength of 482 nm. The activity of Caspase-8 was quantified based on the changes in fluorescence intensity.